T-cell receptor (TCR)-transduced signaling is critical to thymocyte development at the CD4/CD8 double-positive stage, but the molecules involved in this process are not yet fully characterized. We previously demonstrated that GM-CSF/IL-3/IL-5 receptor common β-chain-associated protein (CBAP) modulates ZAP70-mediated T-cell migration and adhesion. On the basis of the high expression of CBAP during thymocyte development, we investigated the function of CBAP in thymocyte development using a CBAP knockout mouse. CBAP-deficient mice showed normal early thymocyte development and positive selection. In contrast, several negative selection models (including TCR transgene, superantigen staphylococcal enterotoxin B, and anti-CD3 antibody treatment) revealed an attenuation of TCR-induced thymocyte deletion in CBAP knockout mice. This phenotype correlated with a reduced accumulation of BIM upon TCR crosslinking in CBAP-deficient thymocytes. Loss of CBAP led to reduced TCR-induced phosphorylation of proteins involved in both proximal and distal signaling events, including ZAP70, LAT, PLCγ1, and JNK1/2. Moreover, TCR-induced association of LAT signalosome components was reduced in CBAP-deficient thymocytes. Our data demonstrate that CBAP is a novel component in the TCR signaling pathway and modulates thymocyte apoptosis during negative selection.

A major mechanism of acquired resistance to immune checkpoint inhibition involves downregulation of antigen presentation, including the MHC I complex itself. Downregulation of antigen presentation on MHC I can render tumor cells invisible to any α/β T cell directed therapy. γδ T cells (γδT) are a small subset of the overall T cell compartment, but are characterized by increased cytolytic capacity relative to α/β T cells. Rather than MHC I, γδT recognize target cells via a complex of heterodimerized butyrophilin (BTN) proteins. Thus, display of BTN heterodimers on the surface of tumor cells may enhance immunity to tumors that have downregulated MHC I, or which express low abundance or low affinity antigens. We have previously reported the generation of a heterodimeric BTN protein targeting the CD19 antigen, referred to as BTN2A1/3A1-Fc-CD19scFv. While the BTN2A1/3A1 complex is a potent activator of γδT expressing the Vγ9δ2 T cell receptor (TCR), which is the major γδT population in human peripheral blood, Vγ9δ2 T cells are not the predominant γδT in many tissues. The murine equivalent of BTN2A1/3A1-Fc-CD19scFv, BTNL1/6-Fc-CD19scFv, stimulated specific proliferation and increased the cytolytic capacity of peripheral blood γδT in mice, but not other tissue-restricted GDT populations. These data suggested that distinct BTN heterodimers may preferentially activate tissue-restricted subsets of GDT. To characterize potential differences between peripheral blood and tissue-restricted γδT , we performed a multi-layered analysis, including single-cell sequencing of γδ TCR from paired peripheral blood and tumor tissues from human cancer patients such as melanoma, prostate and colon cancer. These data identified tissue-specific preferences for individual γδ TCRs, with corresponding tissue-specific preferences for individual BTN proteins. Based on this information, we generated a panel of distinct heterodimeric BTN proteins, and show that specific γδT populations are preferentially activated by specific BTN heterodimers in a lock-and-key fashion. These data are a necessary pre-requisite for designing γδT specific therapeutics that may target both immune neglected and acquired resistant tumors that have limited visibility to α/β T cell directed approaches. Citation Format: Suresh de Silva, George Fromm, Anne Lai, Louis Gonzalez, Arpita Patel, Kyung Jin Yoo, Kellsey Johannes, Kinsley Evans, Keith Wilson, Taylor H. Schreiber. Antigen-specific targeting of tissue-resident gamma delta T cells with recombinant butyrophilin heterodimeric fusion proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1736.