Tumor-associated myeloid cells maintain immunosuppressive microenvironments within tumors. Identification of myeloid-specific receptors to modulate tumor-associated macrophage and myeloid-derived suppressor cell (MDSC) functions remains challenging. The leukocyte immunoglobulin-like receptor B (LILRB) family members are negative regulators of myeloid cell activation. We investigated how LILRB targeting could modulate tumor-associated myeloid cell function. LILRB2 antagonism inhibited receptor-mediated activation of SHP1/2 and enhanced proinflammatory responses. LILRB2 antagonism also inhibited AKT and STAT6 activation in the presence of M-CSF and IL-4. Transcriptome analysis revealed that LILRB2 antagonism altered genes involved in cell cytoskeleton remodeling, lipid/cholesterol metabolism, and endosomal sorting pathways, as well as changed differentiation gene networks associated with inflammatory myeloid cells as opposed to their alternatively activated phenotype. LILRB2 blockade effectively suppressed granulocytic MDSC and Treg infiltration and significantly promoted in vivo antitumor effects of T cell immune checkpoint inhibitors. Furthermore, LILRB2 blockade polarized tumor-infiltrating myeloid cells from non–small cell lung carcinoma tumor tissues toward an inflammatory phenotype. Our studies suggest that LILRB2 can potentially act as a myeloid immune checkpoint by reprogramming tumor-associated myeloid cells and provoking antitumor immunity.

Soluble cytokine receptors function as decoy receptors to attenuate cytokine-mediated signaling and modulate downstream cellular responses. Dysregulated overproduction of soluble receptors can be pathological, such as soluble ST2 (sST2), a prognostic biomarker in cardiovascular diseases, ulcerative colitis, and graft-versus-host disease (GVHD). Although intervention using an ST2 antibody improves survival in murine GVHD models, sST2 is a challenging target for drug development because it binds to IL-33 via an extensive interaction interface. Here, we report the discovery of small-molecule ST2 inhibitors through a combination of high-throughput screening and computational analysis. After in vitro and in vivo toxicity assessment, 3 compounds were selected for evaluation in 2 experimental GVHD models. We show that the most effective compound, iST2-1, reduces plasma sST2 levels, alleviates disease symptoms, improves survival, and maintains graft-versus-leukemia activity. Our data suggest that iST2-1 warrants further optimization to develop treatment for inflammatory diseases mediated by sST2.

Soluble stimulation–2 (ST2) is increased during graft-versus-host disease (GVHD), while Tregs that express ST2 prevent GVHD through unknown mechanisms. Transplantation of Foxp3 ^– T cells and Tregs that were collected and sorted from different Foxp3 reporter mice indicated that in mice that developed GVHD, ST2 ⁺ Tregs were thymus derived and predominantly localized to the intestine. ST2 ^–/– Treg transplantation was associated with reduced total intestinal Treg frequency and activation. ST2 ^–/– versus WT intestinal Treg transcriptomes showed decreased Treg functional markers and, reciprocally, increased Rorc expression. Rorc ^–/– T cells transplantation enhanced the frequency and function of intestinal ST2 ⁺ Tregs and reduced GVHD through decreased gut-infiltrating soluble ST2–producing type 1 and increased IL-4 ^/ IL-10–producing type 2 T cells. Cotransfer of ST2 ⁺ Tregs sorted from Rorc ^–/– mice with WT CD25-depleted T cells decreased GVHD severity and mortality, increased intestinal ST2 ⁺ KLRG1 ⁺ Tregs, and decreased type 1 T cells after transplantation, indicating an intrinsic mechanism. Ex vivo IL-33–stimulated Tregs (Treg _IL-33 ) expressed higher amphiregulin and displayed better immunosuppression, and adoptive transfer prevented GVHD better than control Tregs or Treg _IL-33 cultured with IL-23/IL-17. Amphiregulin blockade by neutralizing antibody in vivo abolished the protective effect of Treg _IL-33 . Our data show that inverse expression of ST2 and RORγt in intestinal Tregs determines GVHD and that Treg _IL-33 has potential as a cellular therapy avenue for preventing GVHD.

Identifying novel cell surface receptors that regulate leukemia cell differentiation and can be targeted to inhibit cellular proliferation is crucial to improve current treatment modalities in acute myeloid leukemia (AML), especially for relapsed or chemotherapy-refractory leukemia. Leukocyte immunoglobulin-like receptor type B (LILRB) is an immunomodulatory receptor originally found to be expressed in myeloid cells. In this study, we found that LILRB receptors can be induced under inflammatory stimuli and chemotherapy treatment conditions. Blockade of LILRB3 inhibited leukemia cell proliferation and leukemia progression. In addition, treatment with LILRB3 blocking antibodies upregulated myeloid lineage differentiation transcription factors, including PU.1, C/EBP family, and IRF, whereas phosphorylation of proliferation regulators, for example, AKT, cyclin D1, and retinoblastoma protein, was decreased. Conversely, transcriptomic analysis showed LILRB3 activation by agonist antibodies may enhance leukemia survival through upregulation of cholesterol metabolism, which has been shown to promote leukemia cell survival. Moreover, LILRB3-targeted CAR T cells exhibited potent antitumor effects both in vitro and in vivo. Taken together, our results suggest that LILRB3 is a potentially potent target for multiple treatment modalities in AML. Significance: LILRB3 regulates differentiation and proliferation in acute myeloid leukemia and can be targeted with monoclonal antibodies and CAR T cells to suppress leukemia growth.