Abstract T cell-centered immunotherapies, including PD-1/PD-L1 blockade, have been successful in treating some types of cancers but not others. The roles of other immune cells including immunosuppressive monocytic cells in tumor development and treatment are not well defined. A better understanding of the molecular regulation of signaling and function of these monocytic cells will support development of novel therapeutic strategies for cancer treatment. The leukocyte Ig-like receptor subfamily B (LILRB) proteins are a group of type I transmembrane glycoproteins with extracellular Ig-like domains that bind ligands and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that can recruit tyrosine phosphatases SHP1, SHP2, or the inositol-phosphatase SHIP. We have been investigating the roles of this family of immune checkpoint receptors in tumor development. We demonstrated that several ITIM-receptors including LILRB2, LILRB4, and a related receptor LAIR1 support leukemia development. As the most restrictively expressed member of the LILRB family, LILRB4 is mainly expressed on monocytic cells but not on stem cells or progenitors. We and others further demonstrated that LILRB4 is a surface marker for monocytic AML and monocytic myeloid-derived suppressor cells. Importantly, we elucidated two novel functions of LILRB4: it supports infiltration of malignant monocytic cells and relieves immune inhibition of T cells. Furthermore, we developed anti-LILRB4 blocking antibodies and CAR-T cells that efficiently inhibit monocytic leukemia development in various mouse models including humanized and patient-derived xenografted mice. Here, based on new preliminary results, we propose the following Aims to test the hypothesis that the expression and signaling of LILRB4 in immunosuppressive monocytic cells support tumor development. In Aim 1, we will determine the function of LILRB4 signaling in immunosupressive monocytic cells and whether LILRB4 blocking/targeting inhibits tumor development. We will then determine how LILRB4 expression is transcriptionally regulated by extrinsic factors in Aim 2. Finally we will determine whether the DNA sensor protein Absent In Melanoma 2 (AIM2) is a key downstream effector of LILRB4 signaling in Aim 3. Our study will provide important mechanistic insights into immune checkpoint biology and directly guide the development of novel immunotherapies for cancer treatment.