VDR^−/− mice have increased HSCs and HPCs in the spleen because of cell-extrinsic effects of VDR loss. (A) Increased HPCs in VDR^−/− spleens. VDR^−/− and VDR^+/+ animals were fed a normal diet immediately after weaning. At 8 weeks of age, animals were killed, and the spleens were removed and crushed through a 40-μm filter. Total splenocyte mononuclear cell (MNC) numbers were obtained with a hemocytometer (trypan blue exclusion). To quantify HPC number, 1 to 2 × 10⁵ splenocytes were plated in M3434 methylcellulose (StemCell Technologies) and scored 12 days later for total colony (defined by a cluster of ≥ 20 cells) number. Colony number was then corrected for total splenocyte number per animal. Bars represent averages; individual mice are represented by individual data points (n = 5). P value was calculated via an unpaired Student t test. (B) Increased HSC frequency in the spleens of VDR^−/− animals. Animals were fed a normal diet immediately after weaning. After death at 8 weeks of age, 2 × 10⁶ spleen MNCs from at least 3 pooled VDR^+/+ or 3 pooled VDR^−/− animals (WT and KO were both CD45.2⁺) were mixed with 2 × 10⁵ BM MNCs from CD45.1⁺ (or CD45.1⁺/CD45.2⁺ double-positive) WT animals and transplanted into 8 to 10 lethally irradiated (9.5 Gy) CD45.1⁺ WT hosts. Sixteen weeks after transplantation, stable engraftment was measured by spleen donor contribution to the PB (obtained by tail vein nicking) in the total MNC fraction and in the B-cell (B220⁺), T-cell (CD4⁺ or CD8⁺), and myeloid (CD11b⁺ or Gr-1⁺) lineages. All antibodies were purchased from BioLegend, BD Biosciences PharMingen, and eBioscience. Antibody clones used are as follows: GK1.5 (anti-CD4), 53-6.7 (anti-CD8a), RA3-6B2 (anti-B220), RB6-865 (anti–Gr-1), and M1/70 (anti-CD11b). Open columns represent data from WT donors; and filled columns, data from KO donors. Error bars represent SE. P values were calculated using Student t tests. (C) No change in total splenocyte numbers. Animals were fed a normal diet immediately after weaning. After death at 8 weeks of age, spleens were removed and crushed through a 40-μm filter. Total splenocyte MNC numbers were obtained with a hemocytometer (trypan blue exclusion). Bars represent averages. Individual mice are represented by individual data points (n = 9). P value was calculated via an unpaired Student t test. (D) No change in splenic HPCs between VDR^−/− and VDR^+/+. VDR^−/− and VDR^+/+ animals were fed a rescue diet immediately after weaning. At 8 weeks of age, animals were killed and analyzed as per the protocol in panel A. Bars represent averages. Individual mice are represented by individual data points (n ≥ 4). P value was calculated via an unpaired Student t test. (E) Little change in HSC frequency after dietary correction. VDR^−/− and VDR^+/+ animals were fed a rescue diet immediately after weaning. At 8 weeks of age, animals were killed, and splenocytes were transplanted according to the protocol in panel B. Sixteen weeks after transplantation, stable engraftment was measured by spleen donor contribution to the PB (obtained by tail vein nicking) in the total MNC fraction and in the B-cell (B220⁺), T-cell (CD4⁺ or CD8⁺), and myeloid (CD11b⁺ or Gr-1⁺) lineages. All antibodies were purchased from BioLegend, BD Biosciences PharMingen, and eBioscience. Antibody clones used are as follows: GK1.5 (anti-CD4), 53-6.7 (anti-CD8a), RA3-6B2 (anti-B220), RB6-865 (anti–Gr-1), and M1/70 (anti-CD11b). Open columns represent data from WT donors; and filled columns, data from KO donors. Error bars represent SE. P values were calculated using Student t tests. (F) No change in total splenocyte numbers. VDR^−/− and VDR^+/+ animals were fed a rescue diet immediately after weaning. After death at 8 weeks of age, spleens were removed and crushed through a 40-μm filter. Total splenocyte MNC numbers were obtained with a hemocytometer (trypan blue exclusion). Bars represent averages. Individual mice are represented by individual data points (n ≥ 8). P value was calculated via an unpaired Student t test.