Fig 2: Genetic loss and pharmacological inhibition of PHGDH impairs GC responses.(A) Mice were injected with sheep RBCs and spleens examined by IHC and flow cytometry 8 days after immunization. (B) Representative flow cytometric analysis of splenic B cells from Phgdh+/+;Cd19-Cre (WT) or Phgdhfl/fl;Cd19-Cre (F/F) before and after immunization to identify GC B cells (CD19+B220+CD38loCD95hi), as well as DZ (CD86loCXCR4hi) and LZ (CD86hiCXCR4lo) B cells, within GC splenic population. (C) Flow cytometric analysis of absolute numbers of B cell subsets within CD19+B220+CD38loCD95hi splenic population from Phgdhfl/fl;Cd19-Cre (n = 6) and Phgdh+/+;Cd19-Cre (n = 4) after immunization. (D) Average GC area (left) and proportion (%) of GC area per spleen area (right) from Phgdhfl/fl;Cd19-Cre (F/F) and Phgdh+/+;Cd19-Cre (WT) mice after immunization. (E) WT mice were immunized with sheep RBCs 1 day before PH-755 treatment (300 mg/kg PH-755 orally twice daily for 7 days). Spleens were analyzed 8 days after immunization. (F) Representative flow cytometric analysis of splenic B cells from mice immunized with sheep RBCs and treated with vehicle/PH-755 to identify GC, DZ, and LZ B cells within GC splenic population. (G) Flow cytometric analysis of absolute number of GC, DZ, and LZ B cells within B220+ splenocytes collected from mice 8 days after sheep RBCs immunization; mice were treated with vehicle (n = 5) or PH-755 (n = 5). (H) Average GCs area (left) and proportion (%) of GC area per spleen area (right) from vehicle- and PH-755–treated mice 8 days after sheep RBC immunization. (I) Summary of NP2-specific plasma cells (PCs; left) and NP2-specific IgG1 PCs (right; total number per popliteal lymph nodes). WT mice were treated with either vehicle (n = 5) or PH-755 (n = 5) for 7 days. Animals were injected with NP-CGG 1 day before PH-755 treatment. Popliteal lymph nodes were collected 8 days after NP-CGG immunization. (J) Serum antibody titers for NP2-specific IgG1 8 days after NP-CGG immunization. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by Mann-Whitney U test (C, G, I, and J) or unpaired t test (D and H).

Fig 3: Genetic loss and pharmacological inhibition of PHGDH reduces lymphoma progression in vivo.(A) Immunoblot of PHGDH, PSAT1, and PSPH expression in splenic B cells from WT (n = 3) and Eµ-Myc mice (n = 6). HSC70 was used as loading control. (B) Representative IHC staining (×20 magnification) for B220, Ki67, MYC, PHGDH, and PSAT1 abundance in sections of spleens from either WT (n = 3) or Eµ-Myc (n = 3) mice. (C) Isotope tracing analysis in splenic B cells isolated from either C57BL/6J WT mice or Eµ-MYC mice and cultured for 2 hours with 13C6-labeled glucose. Serine and glycine levels were measured by LC-MS. The percent distribution of each isotopologue of their respective metabolite pool is represented as mean ± SEM of triplicate cultures and is representative of 3 independent experiments. (D) Schematic showing lymphoma transplantation model, in which Myc/+;Rosa26-CreERT2/+;Phgdhfl/fl lymphoma cells are injected via the tail vein into 9-week-old male C57BL/6J mice. Three days after lymphoma engraftment, mice were randomized to receive either vehicle or tamoxifen treatment by oral gavage for 4 days. Samples were collected 20 days after injection. (E) Representative pictures of spleens from mice (n = 3 per group) sacrificed 20 days after transplantation (left), and quantification of the spleen weight (right). (F) Schematic showing lymphoma transplantation model, in which Myc/+ lymphoma cells are injected via the tail vein into 9-week-old male C57BL/6J mice. Two days after lymphoma engraftment, mice were randomized to be treated with either vehicle or PH-755 by oral gavage for 14 days. (G) Representative pictures of spleens from mice (n = 3 per group) sacrificed after 7 or 14 days after transplantation (left), and quantification of the spleen weight (right). Data are shown as the mean ± SEM. *P < 0.05 and ***P < 0.001, by 2-tailed Student’s t test (E and G).

Fig 4: Loss of the miR-15 family results in B cell progenitor expansion in vitro. (A) Schematic illustration of the sorting strategy for the B cell progenitor in vitro culture. B220+c-KIT+NK1.1-CD19- B cell progenitors were sorted from the bone marrow of miR-15a/bfl/fl Vav-Cre and miR-15a/b+/+ Vav-Cre mice and cultured in the presence of IL-7, FLT3L and SCF. A representative FACS plot depicts the percentages of pro-B (c-KIT+CD25-), intermediate B (c-KIT-CD25-) and pre-B cells (c-KIT-CD25+) under basal conditions after 1 week. (B) Statistical analysis of the distribution of pro-, intermediate and pre-B cell progenitors derived from miR-15a/bfl/fl Vav-Cre mice and miR-15a/b+/+ Vav-Cre mice under basal conditions. (C) Representative FACS blots and statistical analysis of the progenitor B cell differentiation upon IL-7 withdrawal in vitro. (D) Progenitors from miR-15a/bfl/fl Vav-Cre mice and miR-15a/b+/+ Vav-Cre mice were labeled via stable expression of either dsRed or GFP, mixed at equal ratios at a cell density of 5 x 104 cells/ml and tracked over time by flow cytometry in the presence of all growth factors. (E) The bar graph depicts the percentages of EdU+ progenitors derived from miR-15a/bfl/fl Vav-Cre mice or miR-15a/b+/+ Vav-Cre mice after labeling with 10 ug/ml EdU for 4 hrs. (F) Quantification of the percentage of apoptotic progenitors under steady state conditions as analyzed by AnnexinV staining. Error bars depict the standard deviation of the mean. Numbers indicate the percentage of cells in the respective region. MiR-15a/bfl/fl Vav-Cre progenitors were statistically compared to miR-15a/b+/+ Vav-Cre progenitors by an unpaired two-tailed Student’s t-test. *P < 0.05.

Fig 5: OTUB1 deletion in DCs leads to impaired parasite control in toxoplasmosis. a–c OTUB1fl/fl and CD11c-Cre OTUB1fl/fl mice were infected i.p. with 50,000 tachyzoites. Peritoneal cells were isolated from the peritoneal cavities and analyzed by flow cytometry at 2 and 4 days p.i. a Percentages of infected CD11c+ CD8a+, CD11c+ PDCA-1+, and CD11c+ CD11b+ DCs. b Percentages of infected peritoneal CD45+ cells in OTUB1fl/fl and CD11c-Cre OTUB1fl/fl mice. c Percentages of infected peritoneal leukocyte subpopulations in OTUB1fl/fl and CD11c-Cre OTUB1fl/fl mice. Representative FACS plots (upper panel) and statistics (lower panel) are shown (n = 10 for each group). d Mice were infected i.p. with 50,000 tachyzoites for 10 days. Parasite loads in different organs were determined by semiquantitative PCR of tissue DNA (n = 5 for each group). e, f Mice were infected i.p. with three cysts. Parasite load in the brain 30 days p.i. was determined by semiquantitative PCR of tissue DNA (n = 4 for each group) (e). Survival was monitored for 70 days p.i. (f) (n = 10 for each group). Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001; n.s. not significant