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AbstractBackgroundWhile recent clinical trials of combination immunotherapies for hepatocellular carcinoma (HCC) have shown promising clinical efficacy and survival improvements breakthroughs, there is still much room for further improvement. A key limiting factor for HCC immunotherapy is the intrinsic immunosuppression within the liver microenvironment, resulting in suboptimal priming of tumor-specific CD8 cytotoxic T cells and thus immune evasion by the tumor. Hence, identifying new key molecular pathways suppressing T-cell responses within the liver is critical for the rational design of more effective combination immunotherapies for HCC.MethodsWe identified the 5-HT2A serotonin receptor as a potential target for HCC immunotherapy in a chemical screening approach and validated that targeting 5-HT2A signaling could be a viable approach for HCC immunotherapy via in vitro and in vivo studies.ResultsDisruption of 5-HT2A signaling using either a selective antagonist small molecule, ketanserin, or by knockout of its coding gene Htr2a augments the cytotoxic effector phenotype of mouse CD8 T cells activated in vitro with immunosuppressive liver non-parenchymal cells. Ketanserin treatment of in vitro activated human CD8 T cells also increased expression of the cytotoxic effector molecules granzyme B and perforin. Abrogation of 5-HT2A signaling was associated with increased expression of cytotoxicity-related genes such as granzyme B and reduced expression of transcription factors downstream of MAP kinase signaling. In vivo, systemic ketanserin treatment significantly prolonged survival of HCC tumor-bearing mice and was non-inferior to ¦Á-programmed death ligand 1 (PD-L1)+¦Á-vascular endothelial growth factor A (VEGFA) combination antibody treatment. Combining ketanserin with ¦ÁPD-L1+¦ÁVEGFA antibodies also significantly prolonged survival relative to control-treated mice while preserving the occurrence of complete tumor regression observed with ¦ÁPD-L1+¦ÁVEGFA treatment alone.ConclusionsTogether, our data describe a role for 5-HT2A as a negative regulator of the cytotoxic effector phenotype in CD8 T cells and highlight the therapeutic potential of targeting 5-HT2A for HCC immunotherapy.

SummaryAlthough chimeric antigen receptor (CAR) T cells have demonstrated therapeutic activity in hematopoietic malignancies, tumor heterogeneity has impeded the efficacy of CAR T cells and their extension into successful solid tumor treatment. To address these challenges, induced pluripotent stem cell (iPSC)-derived T (iT) cells are engineered to uniformly express CAR and T cell receptor (TCR), enabling targeting of both surface and intracellular antigens, respectively, along with a high-affinity, non-cleavable variant of CD16a (hnCD16) to support antibody-dependent cellular cytotoxicity (ADCC) when combined with therapeutic antibodies. Co-expression of each antitumor strategy on engineered iT cells enables independent and antigen-specific targeting across a diverse set of liquid and solid tumors. In heterogeneous tumor models, coactivation of these modalities is required for measurable antitumor efficacy, with activation of all three modalities displaying maximal efficacy. These data highlight the therapeutic potential of an off-the-shelf engineered iPSC-derived trimodal T cell expressing CAR, TCR, and hnCD16 to combat difficult-to-treat heterogeneous tumors. Graphical abstract Highlights?CAR, TCR, and hnCD16 can be uniformly co-expressed and can function in iT cells?hnCD16 signals through CD3¦Æ and arms iT cells with targeting flexibility through ADCC?Concurring CAR, TCR, and hnCD16 activation demonstrates a cooperative effect?Multi-targeting with trimodal iT cells can control heterogeneous tumors in vivo Yang et al. show that (1) trimodal iPSC cells expressing CAR, TCR, and hnCD16 can commit to T cell lineage, (2) hnCD16 signals through CD3¦Æ in iT cells and arms iT cells with ADCC targeting flexibility, and (3) trimodal iT cells control antigen-heterogeneous tumors in vivo through multi-modal targeting.