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4-1BB causes CAR-T cell death via sequestration of the ubiquitin-modifying enzyme A20

ABSTRACT: CD28 and 4-1BB costimulatory endodomains included in chimeric antigen receptor (CAR) molecules play a critical role in promoting sustained antitumor activity of CAR-T cells. However, the molecular events associated with the ectopic and constitutive display of either CD28 or 4-1BB in CAR-T cells have been only partially explored. In the current study, we demonstrated that 4-1BB incorporated within the CAR leads to cell apoptosis and necroptosis in the absence of CAR tonic signaling. Additionally, we found that cell death induced by 4-1BB co-stimulation is TRAF and RIPK1-dependent. At the molecular level, 4-1BB endodomain causes functional deficiency of the A20 protein in CAR-T cells that appears to be sequestered at the plasma membrane through its binding to the 4-1BB in a TRAF-dependent manner. Genetic modulations of the A20 interplay with 4-1BB obtained by deletion of the TRAF binding motifs of 4-1BB rescue the cell death and enhance the antitumor ability of 4-1BB-costimulated CAR-T cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE267821 | GEO | 2026/05/18

REPOSITORIES: GEO

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Project description:The activation of TLR-MyD88 (Toll like receptor- Myeloid differentiation factor 88) signaling within T cells functions as a potent costimulatory signal that boosts antitumor and antiviral responses. However, the molecular mechanisms underlying the costimulatory processes are poorly understood. We compared microarray gene analysis data between TLR1-TLR2 stimulated and unstimulated T cell receptor transgenic âpmelâ and MyD88-/-pmel CD8+ T cells and identified changes in the expression levels of several TNF family members. In particular, TLR-stimulation increased 4-1BB levels in pmel but not in MyD88-/-pmel T cells. A link between 4-1BB and TLR1-TLR2 signaling in CD8+ T cells was highlighted by in fact that 4-1BB-/- T cells exhibited suboptimal responses to TLR1-TLR2 agonist, but responded normally to CD28 or OX40 costimulation. Moreover, blocking 4-1BB signaling with antibodies also hindered the costimulatory effects of the TLR1-TLR2 agonist. The elevated levels of 4-1BB transcripts in TLR1-TLR2âstimulated cells were not due to increased mRNA stability nor increased histone activation but instead were associated with increased binding of p65 and c-Jun to two distinct 4-1BB promoter sites. Combining TLR1-TLR2 ligand with an agonistic anti-4-1BB antibody enhanced the antitumor activity in mice with established melanoma tumors. These studies reveal that the costimulatory effects of TLR1-TLR2 signaling in CD8+ T cells are in part mediated by 4-1BB and are important for mounting an effective antitumor immune response. CD8+ T cells from the B6.Cg-Thy1/Cy Tg(TcraTcrb)8Rest/J mice, referred to as âpmelâ T cells or from MyD88 knockout pmel mice (MyD88â/âpmel) were sorted. pmel and MyD88â/âpmel T cells were activated using MyD88â/â CD8 T cell-depleted splenocytes pulsed with 10ng/ml of mgp100. This was with or without 10Âµg/ml of Pam3CSK4. pmel or MyD88â/âpmel CD8 T cells were enriched and used for the extraction of RNA used for genomic analysis.

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4-1BB causes CAR-T cell death via sequestration of the ubiquitin-modifying enzyme A20

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