Fig 2: PREX1 facilitates effector T cell differentiation.Naive CD4+ T cells were activated with anti-CD3/anti-CD28 antibody–coated polystyrene beads. (A) RAC1-GTP was measured at indicated times of activation. (B) RAC1-GTP was measured at 5 minutes of activation in control- or PREX1 siRNA–transfected cells from 4 older adults. Statistical analysis was done by 1-tailed, paired Student’s t test. *P < 0.05. (C) RAC1-GTP was measured after 5 minutes of activation in naive CD4+ T cells from 7 young and 7 older adults. Statistical analysis was done by 2-tailed, unpaired Student’s t test. **P < 0.01. (D) Naive CD4+ T cells from 6 older adults were activated for 5 days after control or PREX1 silencing. BLIMP1 and TCF1 expression was measured by flow cytometry. Statistical analysis was done by 2-tailed, paired Student’s t test. **P < 0.01. (E) Schematics of in vivo studies. OT-II naive CD4+ T cells transduced with shCtrl or shPREX1 were injected into B6 mice. Eight days after injection, spleens were harvested and CD4+ T cells were enriched and stained for indicated markers. (F) Frequencies of GFP+CD4+ T cells transduced with shCtrl (red) or shPREX1 (blue) expressing indicated markers (bottom). Five mice per group. (G) Ratio of CD44+CD62L– effector memory (EM) to CD44+CD62L+ central memory (CM) T cells. Five mice per group. Statistical analysis of F and G was done by 2-tailed, unpaired Student’s t test. **P < 0.01.

Fig 3: PREX1 upregulates cell cycle gene programs through facilitating STAT5 nuclear translocation.(A) RAC1-GTP and STAT5 phosphorylation of naive CD4+ T cells from 4 older adults cultured in the presence or absence of IL-7 (5 ng/mL) for 15 minutes. Representative of 2 experiments. Statistical comparison was done by 1-tailed, paired Student’s t test. *P < 0.05. (B) Naive CD4+ T cells from 7 older adults were transfected with either control or PREX1 siRNA and assayed for STAT5 phosphorylation and RAC1-GTP. Statistical comparison was done by 1-tailed, paired Student’s t test. *P < 0.05. (C) Immunofluorescence microscopy images of p-STAT5 (green), nuclei (blue), and merged staining (left panel). Scale bars: 20 μm. Cells from 2 donors were assayed. Data depicted as violin plots showing median and quartiles. Statistical comparison was done for each donor by 2-tailed, unpaired Student’s t test. **P < 0.01. (D) STAT5 ChIP-seq (32) genome tracks of PREX1 and indicated cell cycle genes after IL-2 stimulation. (E) Relative gene expression of PREX1 and cell cycle genes after PREX1 silencing of naive CD4+ T cells from 3 older adults and after 12 days of incubation with IL-7 (5 ng/mL). Statistical comparison was done by 1-tailed, unpaired Student’s t test. *P < 0.05. **P < 0.01.

Fig 4: P-Rex1 expression was correlated with and modulated by Rac2, as opposed to Rac1 or Rac3. The publicly available Kocak database was accessed to evaluate for associations between expression of P-Rex1 and the three Rac effectors: Rac1, Rac2, and Rac3. (A) Whereas Rac1 and Rac3 had statistically significant but relatively weak negative associations with expression of P-Rex1, Rac2 had a robust, significant, and positive correlation with expression of P-Rex1. (B) On Rac GTPase assays, the BE(2)-C/shPREX1 cell line did not demonstrate any appreciable decrease in activation of Rac1 or Rac3 protein relative to BE(2)-C/shCON. Neither neuroblastoma cell line produced any total Rac2 protein.

Fig 5: P-Rex1 was upregulated in pro-metastatic murine models of neuroblastoma. (A) Schematic representation of the in vivo selection model utilized to isolate pro-metastatic, aggressive subclones of neuroblastoma cells from foci of murine hepatic metastases. Image created with BioRender.com. (B) The pro-metastatic subclone, LM2, demonstrated increased normalized linear expression of P-Rex1 on reverse phase protein array analysis relative to the parental BE(2)-C cell line. (C) Representative images demonstrating expression of P-Rex1 in murine hepatic metastases produced after splenic injection of BE(2)-C (left) or LM2 (right) cells using immunohistochemistry. The scale bar represents 100 μm. (D) Murine hepatic metastases produced after splenic injection of the pro-metastatic subclone, LM2, demonstrated increased expression of P-Rex1, relative to murine hepatic metastases produced after splenic injection of the parental cell line, BE(2)-C (p = 0.0012; mean ± SEM.).