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. 2022 May 2;132(9):e153436. doi: 10.1172/JCI153436

Figure 3. Genetic loss and pharmacological inhibition of PHGDH impairs GC responses.

Figure 3

(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).