A Truncated Form of FBXO38 E3 Ligase Downregulates PD-1 Expression in a PD-1 Recombinant 293T Cell Line

Background:

Failure of Chimeric Antigen Receptor (CAR) T-cell therapy for lymphoma and myeloma can be mediated by several mechanisms including T-cell exhaustion (TCE). TCE is a state characterized by loss of proliferative and functional capacity of T-cells. Expression of elevated levels of inhibitory immune checkpoint receptors such as PD-1, LAG-3, TIM-3, TIGIT and CTLA-4 is a hallmark of the exhausted phenotype. Murine models of CAR T-cell therapy show enhanced efficacy when combined with blockade of PD-1 binding to PD-L1. Nevertheless, direct translation to the clinic has not been convincingly demonstrated. However, in lymphoma patients treated with CD19-specific CAR-T cells, PD-1 expression on CD4⁺ CAR-positive cells increased three-fold from infusion to peak blood levels. FBXO38 is an E3 ligase for PD-1 that mediates Lys48-linked polyubiquitination, leading to its subsequent proteasomal degradation via the ubiquitin-proteasome system (UPS). Conditional knockout of Fbxo38 in T cells increases PD-1 surface expression, impairs anti-tumor immunity, and accelerates tumor growth.

Methods:

FBXO38-Flag, FBXO38-Flag mutant (lacking the first 77 amino acids that contain the F-Box domain) were gifted by Dr. Lori Frappier. FBXO38-Flag-GFP (FFG), mutated FBXO38-Flag (MFF), truncated forms of FBXO38-Flag-GFP (1. F3FG residues 1-325, 2. F6FG residues 1-645, and 3. F9FG residues 1-912) and PD1-HA were cloned into the lentiviral vectors pLV-GFP and pLV-mCherry and confirmed by DNA sequencing. Lentivirus of PD1-HA-pLV-GFP and PD1-HA-pLV-mCherry were used to transfect 293T cells. PD1-HA-GFP (PD1G) and PD1-HA-mCherry (PD1CH) cell lines were sorted for GFP and mCherry expression and used for subsequent experiment. The expression and interaction of PD-1, FBXO38, and truncated forms of FBXO38 were interrogated by flow cytometry, Western blot, Immunoprecipitation (IP) and Affinity Purification/Mass Spectrometry (AP-MS).

Results:

FBXO38 (FFG) and the truncated form (F3FG) were shown to interact with PD-1 by IP and AP-MS in PD1CH. Ubiquitinylated PD-1 protein was degraded by the UPS. PD-1 expression was downregulated by FFG and F3FG, but not by MFF, as shown by western blot. Densitometry analysis of western blot data showed that FFG caused a 30% downregulation of intracellular PD-1 protein, whereas F3FG decreased intracellular PD-1 protein by 80% in PD1CH cells. Flow cytometric analysis showed that PD-1 surface expression was decreased by 5% with FFG and 20% with F3F in PD1CH cells. Frappier et al. reported that the USP7 deubiquitinase stabilizes FBXO38 by protecting it from proteasomal degradation. We found that FFG and MFF bound to USP7, while F3FG did not. Both FFG and F3FG bound Skp1, but the ubiquitin domain-mutated MFF did not. Paradoxically F3FG, despite being incapable of binding USP7, decreased intra-cellular PD-1 expression by up to 80%, whereas FFG only showed a 30% reduction. The expression of FFG and F3FG in combination with USP7 did not significantly influence PD-1 expression. Both constructs associated with PD1-HA on immunoprecipitation and MALDI-TOF.

Conclusion:

Full length FBXO38 (FFG) and its truncated form (F3FG) decreased intracellular and membrane-bound PD-1 protein expression in PD1CH cells via the UPS. F3FG appears to be more potent than FFG. USP7 might deubiquitinate FFG and prevent its degradation, enhancing PD-1 protein degradation. However, the truncated form (F3FG) does not associate with USP7 and therefore appears to function independently of USP7. Further study to delineate the mechanism of PD-1 downregulation by F3FG and its effect on CAR-T cell exhaustion is planned.

No relevant conflicts of interest to declare.