CD28 and 4-1{1} 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 4-1{1} in CAR-T cells have been only partially explored.In the current study,we demonstrated that 4-1{1} incorporated within the CAR leads to cell cluster formation and cell death in the forms of both apoptosis and necroptosis in the absence of CAR tonic signaling.Mechanistic studies illustrate that 4-1{1} sequesters A20 to the cell membrane in a TRAF-dependent manner causing A20 functional deficiency that in turn leads to NF-κB hyperactivity,cell aggregation via ICAM-1 overexpression,and cell death including necroptosis via RIPK1/RIPK3/MLKL pathway.Genetic modulations obtained by either overexpressing A20 or releasing A20 from 4-1{1} by deleting the TRAF-binding motifs of 4-1{1} rescue cell cluster formation and cell death and enhance the antitumor ability of 4-1{1}-costimulated CAR-T cells.
4-1{1},a member of the tumor necrosis factor receptor superfamily,is an important co-stimulatory molecule regulating the activity of immune cells across a range of physiological and pathological processes,which culminates in a potent immune response(Figure 1A and B)[1,2].Numerous clinical trials have been conducted utilizing 4–1{1} agonists(Supplemental Table S1);however,previous and ongoing 4-1{1} agonist trials are being conducted without biomarker selection,which potentially explains their modest efficacy.Interestingly,several studies suggest the potential value of utilizing transcriptomics in addition to genomics to identify the unique immunologic signature of individual tumors[3–7].
Chronic Hepatitis B Virus(HBV)infection is a major global health issue leading to liver cirrhosis and hepatocellular carcinoma.Currently available treatments have limited efficacy in completely eradicating the virus.
