We have previously shown that the T-cell protein tyrosine phosphatase (TC-PTP) dephosphorylates the platelet-derived growth factor (PDGF) ␤-receptor. Here, we show that the increased PDGF ␤-receptor phosphorylation in TC-PTP knockout (ko) mouse embryonic fibroblasts (MEFs) occurs primarily on the cell surface. The increased phosphorylation is accompanied by a TC-PTP-dependent, monensin-sensitive delay in clearance of cell surface PDGF ␤-receptors and delayed receptor degradation, suggesting PDGF ␤-receptor recycling. Recycled receptors could also be directly detected on the cell surface of TC-PTP ko MEFs. The effect of TC-PTP depletion was specific for the PDGF ␤-receptor, because PDGF ␣-receptor homodimers were cleared from the cell surface at the same rate in TC-PTP ko MEFs as in wild-type MEFs. Interestingly, PDGF ␣␤-receptor heterodimers were recycling. Analysis by confocal microscopy revealed that, in TC-PTP ko MEFs, activated PDGF ␤-receptors colocalized with Rab4a, a marker for rapid recycling. In accordance with this, transient expression of a dominant-negative Rab4a construct increased the rate of clearance of cell surface receptors on TC-PTP ko MEFs. Thus, loss of TC-PTP specifically redirects the PDGF ␤-receptor toward rapid recycling, which is the first evidence of differential trafficking of PDGF receptor family members.

B-cell malignancies upregulate the B-cell lymphoma 2 (Bcl-2) family inhibitors of the intrinsic apoptosis pathway, making them therapy resistant. However, small-molecule inhibitors of Bcl-2 family members such as ABT-737 restore a functional apoptosis pathway in cancer cells, and its oral analog ABT-263 (Navitoclax) has entered clinical trials. Gene engineered chimeric antigen receptor (CAR) T cells also show promise in B-cell malignancy, and as they induce apoptosis via the extrinsic pathway, we hypothesized that small-molecule inhibitors of the Bcl-2 family may potentiate the efficacy of CAR T cells by engaging both apoptosis pathways. CAR T cells targeting CD19 were generated from healthy donors as well as from pre-B-ALL (precursor-B acute lymphoblastic leukemia) patients and tested together with ABT-737 to evaluate apoptosis induction in five B-cell tumor cell lines. The CAR T cells were effective even if the cell lines exhibited different apoptosis resistance profiles, as shown by analyzing the expression of apoptosis inhibitors by PCR and western blot. When combining T-cell and ABT-737 therapy simultaneously, or with ABT-737 as a presensitizer, tumor cell apoptosis was significantly increased. In conclusion, the apoptosis inducer ABT-737 enhanced the efficacy of CAR T cells and could be an interesting drug candidate to potentiate T-cell therapy.

Previous studies showed that loss of the T-cell protein tyrosine phosphatase (TC-PTP) induces Rab4a-dependent recycling of the platelet-derived growth factor (PDGF) ␤-receptor in mouse embryonic fibroblasts (MEFs). Here we identify protein kinase C (PKC) ␣ as the critical signaling component that regulates the sorting of the PDGF ␤-receptor at the early endosomes. Down-regulation of PKC abrogated receptor recycling by preventing the sorting of the activated receptor into EGFP-Rab4a positive domains on the early endosomes. This effect was mimicked by inhibition of PKC␣, using myristoylated inhibitory peptides or by knockdown of PKC␣ with shRNAi. In wt MEFs, short-term preactivation of PKC by PMA caused a ligand-induced PDGF ␤-receptor recycling that was dependent on Rab4a function. Together, these observations demonstrate that PKC activity is necessary for recycling of ligand-stimulated PDGF ␤-receptor to occur. The sorting also required Rab4a function as it was prevented by expression of EGFP-Rab4aS22N. Preventing receptor sorting into recycling endosomes increased the rate of receptor degradation, indicating that the sorting of activated receptors at early endosomes directly regulates the duration of receptor signaling. Activation of PKC through the LPA receptor also induced PDGF ␤-receptor recycling and potentiated the chemotactic response to PDGF-BB. Taken together, our present findings indicate that sorting of PDGF ␤-receptors on early endosomes is regulated by sequential activation of PKC␣ and Rab4a and that this sorting step could constitute a point of cross-talk with other receptors.