Enhanced T cell effector activity by targeting the Mediator kinase moduleT cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were MED12 and CCNC , components of the Mediator kinase module. Targeted MED12 deletion enhanced antitumor activity and sustained the effector phenotype in CAR- and T cell receptor–engineered T cells, and inhibition of CDK8/19 kinase activity increased expansion of nonengineered T cells. MED12 -deficient T cells manifested increased core Meditator chromatin occupancy at transcriptionally active enhancers—most notably for STAT and AP-1 transcription factors—and increased IL2RA expression and interleukin-2 sensitivity. These results implicate Mediator in T cell effector programming and identify the kinase module as a target for enhancing potency of antitumor T cell responses.
Engineered CD47 protects T cells for enhanced antitumour immunityAbstract Adoptively transferred T cells and agents designed to block the CD47–SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system 1,2 . Here we administered anti-CD47 antibodies in combination with adoptively transferred T cells with the goal of enhancing antitumour efficacy but observed abrogated therapeutic benefit due to rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors. Anti-CD47-antibody-mediated CAR T cell clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered the CD47 variant CD47(Q31P) (47 E ), which engages SIRPα and provides a ‘don’t eat me’ signal that is not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47 E are resistant to clearance by macrophages after treatment with anti-CD47 antibodies, and mediate substantial, sustained macrophage recruitment to the tumour microenvironment. Although many of the recruited macrophages manifested an M2-like profile 3 , the combined therapy synergistically enhanced antitumour efficacy. Our study identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T-cell-directed therapeutics with those designed to activate macrophages. It delivers a therapeutic approach that is capable of simultaneously harnessing the antitumour effects of T cells and macrophages, offering enhanced potency against solid tumours.
A versatile CRISPR-Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cellsSkin basal cell carcinomas assemble a pro-tumorigenic spatially organized and self-propagating Trem2+ myeloid nicheAbstract Cancer immunotherapies have revolutionized treatment but have shown limited success as single-agent therapies highlighting the need to understand the origin, assembly, and dynamics of heterogeneous tumor immune niches. Here, we use single-cell and imaging-based spatial analysis to elucidate three microenvironmental neighborhoods surrounding the heterogeneous basal cell carcinoma tumor epithelia. Within the highly proliferative neighborhood, we find that TREM2 + skin cancer-associated macrophages (SCAMs) support the proliferation of a distinct tumor epithelial population through an immunosuppression-independent manner via oncostatin-M/JAK-STAT3 signaling. SCAMs represent a unique tumor-specific TREM2 + population defined by VCAM1 surface expression that is not found in normal homeostatic skin or during wound healing. Furthermore, SCAMs actively proliferate and self-propagate through multiple serial tumor passages, indicating long-term potential. The tumor rapidly drives SCAM differentiation, with intratumoral injections sufficient to instruct naive bone marrow-derived monocytes to polarize within days. This work provides mechanistic insights into direct tumor-immune niche dynamics independent of immunosuppression, providing the basis for potential combination tumor therapies.
Cell-autonomous control of CAR signaling and receptor shedding via ADAM17-mediated proteolysisWe sought to endow T cell autonomous regulation of cell surface protein expression by exploiting the conditional proteolytic activity of ADAM17 following T cell activation. Screening of canonical ADAM17 substrates yielded a minimal 15-aa CD62L-derived motif that confers rapid and reversible cleavage of a receptor following T cell activation-termed activation-induced release (AIR). Embedding AIR into tonic-signaling CARs reduced basal CAR expression proportional to the degree of tonic signaling induced, curtailing exhaustion and improving antitumor potency. In non-tonic signaling CARs, AIR decreased activation-induced cell death and enhanced T cell expansion after stimulation. AIR's modularity supports higher-order logic-gating; AIR-regulated peptide masks enable antigen-dependent unmasking of an EGFR-targeting CAR. Finally, CRISPR knockin of AIR into endogenous FAS or TGFBR2 endowed them with activation-induced shedding, which enhanced tumor clearance while preserving signaling in non-activating conditions. AIR is a compact switch that provides fast, autonomous regulation of surface proteins for next-generation cell therapies.