Faculty Bibliography

The outbreak of coronavirus disease 2019 (COVID-19) is an unprecedented global health crisis. Tissue and peripheral blood analysis indicate profound, aberrant myeloid cell activation, cytokine storm, and lymphopenia, with unknown immunopathological mechanisms. Spatiotemporal control of the quality and quantity of the antiviral immune responses involves synchronized cellular and molecular cascades and cross-talk between innate and adaptive immunity. Dysregulated responses in immunity, such as at the stages of immune sensing, alarming, polarization, and resolution, may contribute to disease pathology. Herein, we approach SARS-CoV-2 through an immunomodulatory lens, discussing possible mechanisms of the asynchronized antiviral immune response and proposing potential therapeutic strategies to correct the dysregulation.

Cytokines were the first modern immunotherapies to produce durable responses in patients with advanced cancer, but they have only modest efficacy and limited tolerability^1,2. In an effort to identify alternative cytokine pathways for immunotherapy, we found that components of the interleukin-18 (IL-18) pathway are upregulated on tumour-infiltrating lymphocytes, suggesting that IL-18 therapy could enhance anti-tumour immunity. However, recombinant IL-18 previously did not demonstrate efficacy in clinical trials³. Here we show that IL-18BP, a high-affinity IL-18 decoy receptor, is frequently upregulated in diverse human and mouse tumours and limits the anti-tumour activity of IL-18 in mice. Using directed evolution, we engineered a 'decoy-resistant' IL-18 (DR-18) that maintains signalling potential but is impervious to inhibition by IL-18BP. Unlike wild-type IL-18, DR-18 exerted potent anti-tumour effects in mouse tumour models by promoting the development of poly-functional effector CD8⁺ T cells, decreasing the prevalence of exhausted CD8⁺ T cells that express the transcriptional regulator of exhaustion TOX, and expanding the pool of stem-like TCF1⁺ precursor CD8⁺ T cells. DR-18 also enhanced the activity and maturation of natural killer cells to effectively treat anti-PD-1 resistant tumours that have lost surface expression of major histocompatibility complex class I molecules. These results highlight the potential of the IL-18 pathway for immunotherapeutic intervention and implicate IL-18BP as a major therapeutic barrier.

PURPOSE/OBJECTIVE:Multiple negative regulators restrict the ability of T cells to attack tumors. This work demonstrates the role of PI3K-interacting protein 1 (Pik3ip1) in restraining T-cell responses and antitumor immunity. EXPERIMENTAL DESIGN/METHODS:mice and a Pik3ip1 fusion protein were generated to investigate the effect of Pik3ip1 on T-cell-mediated antitumor immunity in MC38 and B16-F10 tumor models. Immunoblotting and confocal microscopy were used to identify inhibitory effects of Pik3ip1 on T-cell receptor (TCR) signaling. Pik3ip1 expression was quantified, and its impact on T-cell function in human tumors was measured. RESULTS:mice exhibited a marked increase in antitumor immunity and were resistant to tumor growth. Furthermore, Pik3ip1 extracellular domain fusion protein enhanced MC38 tumor growth was observed. Mechanistically, we found that Pik3ip1 inhibited TCR signaling by mediating the degradation of SLP76 through Pik3ip1 oligomerization via its extracellular region. Consistent with the results from the mouse models, PIK3IP1 expression correlated with T-cell dysfunction in human tumors. CONCLUSIONS:Our data reveal a critical role for Pik3ip1 as a novel inhibitory immune regulator of T-cell responses and provide a potential molecular target for cancer immunotherapy.

4-1BB (CD137) is an important costimulatory molecule upregulated on antigen-experienced T cells, however, clinical development of 4-1BB agonists has stalled because of significant liver immuno-toxicity. Using rational protein engineering, a next-generation anticalin-antibody-based therapy achieved localized 4-1BB activation triggered by tumor-expressed antigen, helping to revitalize this pathway in immuno-oncology.See related article by Hinner et al., p. 5878.

PURPOSE/OBJECTIVE:To determine the tumor tissue/cell distribution, functional associations and clinical significance of PD-1, LAG-3 and TIM-3 in human non-small-cell lung cancer (NSCLC). EXPERIMENTAL DESIGN/METHODS:Using multiplexed quantitative immunofluorescence (QIF) we measured CD3, PD-1, LAG-3 and TIM-3 in >800 NSCLCs from three tissuemicroarray-based cohorts. Associations between markers and tumor genomics were studied in TCGA-NSCLC dataset. Using mass-cytometry (CyTOF) analysis from 20 resected NSCLCs, we determined the levels, co-expression and functional profile of PD-1, LAG-3 and TIM-3 expressing immune cells. Finally, we measured the markers in 90 NSCLCs from patients treated with PD-1 axis blockers. RESULTS:PD-1, LAG-3 and TIM-3 were detected in TILs from 55%, 41.5% and 25.3% of cases, respectively. These markers showed association with each other, but not with clinicopathologic variables and survival in cases without immunotherapy. The markers were lower in EGFR-mutated adenocarcinomas and partially associated with tumor-mutational burden. In single-cell CyTOF analysis, PD-1 and LAG-3 were predominantly localized on T/NKT-cells; while TIM-3 was higher in NK-cells and macrophages. Co-expression of PD-1,LAG-3 and TIM-3 was associated with T-cell activation, effector function and proliferation, but also with pro-apoptotic markers. LAG-3 and TIM-3 were present in TILs lacking PD-1 and elevated baseline LAG-3 was associated with shorter progression-free survival after PD-1 axis blockade. CONCLUSIONS:PD-1, LAG-3 and TIM-3 have distinct tissue/cell distribution, functional implications and genomic correlates in NSCLC. Expression of these receptors is associated with activation, but also with pro-apoptotic T-cell phenotype. Elevated LAG-3 is associated with insensitivity to PD-1 blockade suggesting independence of these immune evasion pathways.

Macrophages have been suggested to contribute to constructing a cancer stem cell (CSC) niche. However, whether and how macrophages regulate the activity of CSCs through juxtacrine signaling are poorly understood. Here we report LSECtin, a transmembrane protein highly expressed on tumor-associated macrophages (TAMs), enhances stemness of breast cancer cells (BCCs). We identified BTN3A3, a B7 family member with previously unknown functions as the receptor for LSECtin on BCCs responsible for stemness-promoting effect of LSECtin. In mice bearing human tumor xenografts, either macrophage-specific ablation of LSECtin or silencing of BTN3A3 in BCCs decreased CSC frequency and tumor growth. Admixture of LSECtin-positive macrophages increased the tumorigenic activity of BCCs dependent on BTN3A3. Disruption of the LSECtin-BTN3A3 axis with BTN3A3-Fc or anti-BTN3A3 mAb has a therapeutic effect on breast cancer. These findings define a juxtacrine signaling mechanism by which TAMs promote cancer stemness. Targeting this axis in the CSC niche may provide potential therapies to breast cancer.

Enterovirus D68 (EV-D68) is an atypical nonpolio enterovirus that mainly infects the respiratory system of humans, leading to moderate-to-severe respiratory diseases. In rare cases, EV-D68 can spread to the central nervous system and cause paralysis in infected patients, especially young children and immunocompromised individuals. There is currently no approved vaccine or antiviral available for the prevention and treatment of EV-D68. In this study, we aimed to improve the antiviral potency and selectivity of a previously reported EV-D68 inhibitor, dibucaine, through structure-activity relationship studies. In total, 60 compounds were synthesized and tested against EV-D68 using the viral cytopathic effect assay. Three compounds 10a, 12a, and 12c were identified to have significantly improved potency (EC₅₀ < 1 μM) and a high selectivity index (>180) compared with dibucaine against five different strains of EV-D68 viruses. These compounds also showed potent antiviral activity in neuronal cells, such as A172 and SH-SY5Y cells, suggesting they might be further developed for the treatment of both respiratory infection as well as neuronal infection.

Overexpression of the B7-H1 (PD-L1) molecule in the tumor microenvironment (TME) is a major immune evasion mechanism in some patients with cancer, and antibody blockade of the B7-H1/PD-1 interaction can normalize compromised immunity without excessive side-effects. Using a genome-scale T cell activity array, we identified Siglec-15 as a critical immune suppressor. While only expressed on some myeloid cells normally, Siglec-15 is broadly upregulated on human cancer cells and tumor-infiltrating myeloid cells, and its expression is mutually exclusive to B7-H1, partially due to its induction by macrophage colony-stimulating factor and downregulation by IFN-γ. We demonstrate that Siglec-15 suppresses antigen-specific T cell responses in vitro and in vivo. Genetic ablation or antibody blockade of Siglec-15 amplifies anti-tumor immunity in the TME and inhibits tumor growth in some mouse models. Taken together, our results support Siglec-15 as a potential target for normalization cancer immunotherapy.

Lymphocyte-activation gene 3 (LAG-3) is an immune inhibitory receptor, with major histocompatibility complex class II (MHC-II) as a canonical ligand. However, it remains controversial whether MHC-II is solely responsible for the inhibitory function of LAG-3. Here, we demonstrate that fibrinogen-like protein 1 (FGL1), a liver-secreted protein, is a major LAG-3 functional ligand independent from MHC-II. FGL1 inhibits antigen-specific T cell activation, and ablation of FGL1 in mice promotes T cell immunity. Blockade of the FGL1-LAG-3 interaction by monoclonal antibodies stimulates tumor immunity and is therapeutic against established mouse tumors in a receptor-ligand inter-dependent manner. FGL1 is highly produced by human cancer cells, and elevated FGL1 in the plasma of cancer patients is associated with a poor prognosis and resistance to anti-PD-1/B7-H1 therapy. Our findings reveal an immune evasion mechanism and have implications for the design of cancer immunotherapy.

4-1BB (CD137, tumor necrosis factor receptor superfamily 9) is an inducible costimulatory receptor expressed on activated T and natural killer (NK) cells. 4-1BB ligation on T cells triggers a signaling cascade that results in upregulation of antiapoptotic molecules, cytokine secretion, and enhanced effector function. In dysfunctional T cells that have a decreased cytotoxic capacity, 4-1BB ligation demonstrates a potent ability to restore effector functions. On NK cells, 4-1BB signaling can increase antibody-dependent cell-mediated cytotoxicity. Agonistic monoclonal antibodies targeting 4-1BB have been developed to harness 4-1BB signaling for cancer immunotherapy. Preclinical results in a variety of induced and spontaneous tumor models suggest that targeting 4-1BB with agonist antibodies can lead to tumor clearance and durable antitumor immunity. Clinical trials of 2 agonist antibodies, urelumab and utomilumab, are ongoing. Despite initial signs of efficacy, clinical development of urelumab has been hampered by inflammatory liver toxicity at doses >1 mg/kg. Utomilumab has a superior safety profile, but is a less potent 4-1BB agonist relative to urelumab. Both antibodies have demonstrated promising results in patients with lymphoma and are being tested in combination therapy trials with other immunomodulatory agents. In an effort to optimally leverage 4-1BB-mediated immune activation, the next generation of 4-1BB targeting strategies attempts to decouple the observed antitumor efficacy from the on-target liver toxicity. Multiple therapeutics that attempt to restrict 4-1BB agonism to the tumor microenvironment and minimize systemic exposure have emerged. 4-1BB is a compelling target for cancer immunotherapy and future agents show great promise for achieving potent immune activation while avoiding limiting immune-related adverse events.