HSC numbers are normal in steady-state adult BM of Tnfrsf1-dKO mice. (a) In six separate experiments with three mice in each experiment, BM cells from two tibiae and two femurs per mouse were individually isolated from age (8–12 wk old)- and sex-matched WT (n = 18) and Tnfrsf1-dKO (n = 18) mice, and mean (SD) cellularities were determined. (b and c) In two separate experiments, age (8–12 wk old)- and sex-matched adult WT C57BL/6 and Tnfrsf1-dKO mice, five of each genotype, were analyzed individually for frequencies of LSKFLT3⁻ cells by FACS. (b) Representative FACS analysis for each of the genotypes. Displayed percentages of population frequencies of total BM cells are mean values for five mice in each group, each mouse analyzed individually. (c) BM cell numbers and absolute numbers of LSKFLT3⁻ cells were determined for each mouse individually (see Materials and methods). Mean (SD) values of five mice are shown. (d) In two separate experiments with 14 recipients in each, 20,000 unfractionated BM cells from either WT (CD45.1⁺/2⁺) or Tnfrsf1-dKO (CD45.1⁻/2⁺) mice were transplanted, together with 200,000 competitor BM cells (CD45.1⁺/2⁻), into lethally irradiated congenic WT recipients (CD45.1⁺/2⁻). 16 wk after transplantation, recipients were analyzed individually for multilineage reconstitution. The percentage of PB donor myeloid reconstitution in positively reconstituted mice is indicated. Horizontal lines represent median reconstitution levels. The frequencies of reconstituted mice (as defined in Materials and methods) out of total transplanted mice are indicated. The indicated p-value is for difference in mean donor reconstitution levels of mice transplanted with Tnfrsf1-dKO and WT BM cells. (e and f) CD45.1⁺ BM cells from four WT mice and CD45.2⁺ BM cells from four Tnfrsf1-dKO mice were individually isolated, and subsequently cells from each of the WT replicates were mixed with cells from one of the Tnfrsf1-dKO mice. Cells from each genotype were mixed at equal numbers, and each cell mixture was analyzed individually. Cell mixtures were analyzed by FACS for G0 (DAPI^low/Ki67⁻), G1 (DAPI^low/Ki67⁺), and S/G2/M (DAPI^high/Ki67⁺) cell cycle stages within the LSKFLT3⁻ HSC compartment. Percentages represent mean (SD) values of all mice.

TNF-mediated suppression in vitro is critically dependent on expression of both TNF receptors. (a) In four separate experiments, with two replicates in each experiment, 20,000 whole BM cells from 8–12-wk-old WT, Tnfrsf1-dKO, Tnfrsf1a^−/−, and Tnfrsf1b^−/− mice (all backcrossed for 10 generations with C57BL/6 mice) were plated in methylcellulose supplemented with G-CSF and SCF in the presence and absence of TNF (±TNF). CFU-GM was scored after 7 d in culture. Mean (SD) values for all mice and experiments are shown. (b and c) 100,000 whole BM cells/dish were seeded in semisolid methylcellulose medium supplemented with SCF, erythropoietin, and thrombopoietin, ±TNF, and analyzed for erythroid (BFU-E) and megakaryocyte (CFU-Meg) colony potentials after 8 d in culture. Mean (SD) data from four methylcellulose replicates in two separate experiments are shown. (d) FACS-purified LSK cells from pooled mice of each indicated genotype were single cell sorted into Terasaki plates containing SCF and G-CSF, ±TNF. After 11 d of culture, the number of colonies (>50 cells) was scored. Mean (SD) values from two experiments with two replicate plates each and each plate containing 60 cells are shown.

Enhanced activity of TNF receptor–deficient HSCs after transplantation. (a–f) BM cells were pooled from three Tnfrsf1-dKO (CD45.2⁺) or three WT (CD45.1⁺) mice (8–12 wk old), and for each of the genotypes 10⁶ unfractionated BM cells were competitively cotransplanted into lethally irradiated congenic WT recipients (CD45.1⁺/2⁺). Recipient mice were evaluated for PB multilineage reconstitution levels at the indicated time points. (a) Representative FACS analysis illustrating PB myeloid chimerism evaluation. (b) PB myeloid reconstitution derived from transplanted Tnfrsf1-dKO and WT cells was analyzed at the indicated time points. At the time points indicated by the vertical dashed lines, mice were sacrificed, and 0.5 femur equivalent of BM cells were serially transplanted into newly irradiated (CD45.1⁺/2⁺) recipients. Displayed are PB myeloid chimerism levels. Results are mean (SEM) values from six separate experiments with five to seven recipients in each experiment. (c–f) 16 wk after the primary transplantation, PB analysis of T (CD4/CD8), myeloid (MAC1), and B (B220) cell reconstitution was performed (d), BM was analyzed for LSK chimerism (f), and mean percentages for all recipients are presented. (c and e) Representative FACS analysis for reconstitution levels within the PB and BM are shown. (d and f) Results are from six separate experiments with five to seven recipients in each experiment. Mean (SEM) values are shown. (g–j) BM cells from 8–12 wk old Tnfrsf1a^−/− (g and i) and Tnfrsf1b^−/− (h and j) mice (CD45.2⁺, backcrossed for 10 generations with C57BL/6 mice) were transplanted in competition with WT BM cells (CD45.1⁺) into lethally irradiated WT (CD45.1⁺/2⁺) recipients. PB myeloid chimerism levels (g and h) and BM LSK chimerism levels (i and j) at the indicated time points after primary and secondary (indicated by dashed line) transplantations are shown. All results are mean (SEM) values from two separate experiments with five to seven recipients in each experiment. PB and BM reconstitution analysis were performed as illustrated in panels a and e, respectively.

In vivo administration of TNF suppresses cycling HSCs. (a) Experimental design. Age (8–12 wk old)- and sex-matched C57BL/6 WT mice received 150 mg/kg 5-FU or PBS 3 d before intravenous injections of 3 × 2 µg TNF or PBS. (b) BM cellularity in mice treated with PBS, PBS and TNF, 5-FU, or 5-FU and TNF. Mean (SD) values of three experiments, each with three individual mice in each treatment group, are shown. (c–e) At day 0, 1/50 of unfractionated BM cells (CD45.2⁺) in two femurs and two tibias from treated mice in each of the indicated groups were transplanted in competition with 10⁶ unfractionated WT BM (CD45.1⁺) cells into congenic lethally irradiated WT recipients (CD45.1⁺/2⁺). 16 wk after transplantation, PB reconstitution levels were analyzed for percentage of CD45.2⁺ contribution within the T cell (c), B cell (d), and myeloid blood cell (e) lineages. (f) Bars show calculated numbers (see Materials and methods) of total CRUs in PBS-, PBS + TNF–, 5-FU–, and 5-FU + TNF–treated donor BM cells based on PB levels of myeloid chimerism 16 wk after transplantation. (c–f) Results are mean (SD) values from three separate experiments, each with five to seven recipients/group.