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Supplemental Figures 1-8 plus Legends

There is a strong correlation between myeloid-derived suppressor cells (MDSC) and resistance to immune checkpoint blockade (ICB), but the detailed mechanisms underlying this correlation are largely unknown. Using single-cell RNA sequencing analysis in a bilateral tumor model, we found that immunosuppressive myeloid cells with characteristics of fatty acid oxidative metabolism dominate the immune-cell landscape in ICB-resistant subjects. In addition, we uncovered a previously underappreciated role of a serine/threonine kinase, PIM1, in regulating lipid oxidative metabolism via PPARγ-mediated activities. Enforced PPARγ expression sufficiently rescued metabolic and functional defects of Pim1−/− MDSCs. Consistent with this, pharmacologic inhibition of PIM kinase by AZD1208 treatment significantly disrupted the myeloid cell–mediated immunosuppressive microenvironment and unleashed CD8+ T-cell–mediated antitumor immunity, which enhanced PD-L1 blockade in preclinical cancer models. PIM kinase inhibition also sensitized nonresponders to PD-L1 blockade by selectively targeting suppressive myeloid cells. Overall, we have identified PIM1 as a metabolic modulator in MDSCs that is associated with ICB resistance and can be therapeutically targeted to overcome ICB resistance.

Supplementary Table 1

There is a strong correlation between myeloid-derived suppressor cells (MDSC) and resistance to immune checkpoint blockade (ICB), but the detailed mechanisms underlying this correlation are largely unknown. Using single-cell RNA sequencing analysis in a bilateral tumor model, we found that immunosuppressive myeloid cells with characteristics of fatty acid oxidative metabolism dominate the immune-cell landscape in ICB-resistant subjects. In addition, we uncovered a previously underappreciated role of a serine/threonine kinase, PIM1, in regulating lipid oxidative metabolism via PPARγ-mediated activities. Enforced PPARγ expression sufficiently rescued metabolic and functional defects of Pim1−/− MDSCs. Consistent with this, pharmacologic inhibition of PIM kinase by AZD1208 treatment significantly disrupted the myeloid cell–mediated immunosuppressive microenvironment and unleashed CD8+ T-cell–mediated antitumor immunity, which enhanced PD-L1 blockade in preclinical cancer models. PIM kinase inhibition also sensitized nonresponders to PD-L1 blockade by selectively targeting suppressive myeloid cells. Overall, we have identified PIM1 as a metabolic modulator in MDSCs that is associated with ICB resistance and can be therapeutically targeted to overcome ICB resistance.

Macrophages can impact the development of tumors and their responses to therapy. However, we lack a full understanding of how macrophage activation and survival are regulated and might be therapeutically modulated. To address these questions, we turned to a host-pathogen interaction that is likely to have impacted the evolution of these processes: infection by Mycobacterium tuberculosis (Mtb), the leading cause of death from infection. Once internalized by macrophages, Mtb resists killing by macrophages and replicates within them. Ultimately, Mtb infection results in death of macrophages, allowing dissemination of the pathogen to other cells. To explore how Mtb induces macrophage cell death and how macrophage cell death might impact host defense against Mtb, we performed a genome-wide CRISPR-Cas9 recessive genetic screen in RAW264.7 macrophages. We discovered that the absence of components of the type I interferon signaling pathway, including the IFN-α/β receptor (IFNAR), delays Mtb-induced cell death. It is known that Mtb infection induces macrophages to produce type I interferon. Our finding directly links type I interferon signaling pathway and Mtb-induced macrophage death. We are currently working on the mechanism of the type I IFN-induced death of bacterially-stimulated macrophages. Meanwhile, we are testing blockers of this pathway as host-directed therapy against TB and have seen striking protective effects of anti-IFNAR1 mAb in Mtb-infected mice, whether the mAb is administered before or after infection. Further exploration of the protective effect of anti-IFNAR mAb could point to an antibody-based, host-directed therapy. Acknowledgment: We thank Prof. R. Schreiber, Washington University, for facilitating access to anti-IFNAR mAb. Citation Format: Ryan Zander, David Schauder, Gang Xin, Christine Nguyen, Xiaopeng Wu, Weiguo Cui. Exploration and exploitation of macrophage death pathways: Infection by Mycobacterium tuberculosis as a model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B201.

During chronic viral infection and cancer, CD8 T-cells undergo a differentiation process commonly referred to as T-cell exhaustion. This process is traditionally defined by a stepwise loss of effector functions, eventually leading to cell death. Despite their inability to completely clear the infection, exhausted T-cells are still necessary for limiting viral replication during infection. Thus, it has been proposed that functional adaptation is a more appropriate term for T-cell exhaustion, as CD8 T-cells may be undergoing a multifaceted process of differentiation to better meet the needs of a chronic infection. In line with this hypothesis, it has recently been demonstrated that CD8 T-cells responding to chronic infection are non-homogenous and can be compartmentalized into at least two major subsets, with a TCF-1+ subset serving as a progenitor population that can give rise to a more terminally exhausted TCF-1- subset. However, whether additional heterogeneity exists among CD8 T-cells responding to persistent infection remains unclear. Here, we used ScRNA-seq to fully characterize the heterogeneity of CD8 T-cells during chronic LCMV Cl13 infection. We identified that several transcriptionally distinct subsets of CD8 T-cells develop during chronic LCMV infection, with 3 particular clusters, Slamf6, Pdcd1, and Cx3cr1 cell subsets dominating the antiviral CD8 T-cell response. Importantly both ScRNA-seq and flow cytometric analyses demonstrated that differential expression of cell surface receptors CX3CR1 and Ly108 (encoded by Slamf6) can distinguish these 3 major T-cell subsets. Notably, Ly108 cells shared similar characteristics to the previously described progenitor population and displayed elevated expression of TCF-1. Conversely, CX3CR1 CD8 T-cells displayed increased expression of killer cell lectin-like receptors Klre1 and Klra9, and the TFs T-bet and Zeb2, whereas CX3CR1-Ly108- (DN) cells exhibited elevated expression of multiple co-inhibitory receptors and the TFs Eomes and Nr4a2. Ex vivo functional analyses further indicated that Ly108 CD8 T-cells exhibit an enhanced capacity to co-produce IFN-γ and TNF-α upon GP33 peptide stimulation, whereas CX3CR1 CD8 T-cells display augmented cytotoxicity against peptide-pulsed targeT-cells. Sc trajectory modeling using Monocle analyses predicted that Ly108 CD8 T-cells give rise to both CX3CR1 and DN subsets, with the DN subset branch appearing closer in pseudotime to the Ly108 progenitor subset. To determine the in vivo differentiation trajectory, proliferative potential, and phenotypic stability of these 3 subsets, we performed adoptive transfer experiments using congenically marked CD8 T-cells. Importantly, and consistent with our Monocle predictions, our results demonstrate that Ly108 CD8 T-cells display robust secondary proliferation and give rise to both CX3CR1 and DN subsets. By contrast, CX3CR1 cells retained high CX3CR1 and T-bet expression and did not differentiate into Ly108 or DN CD8 T-cells. Intriguingly, although the DN subset appeared to be the most phenotypically and functionally exhausted subset, more than half of their progeny acquired high CX3CR1 and T-bet expression, indicating that this subset may not be as terminally differentiated as its CX3CR1 counterpart. Notably, our ScRNA-seq analyses also identified that DN cells displayed the highest levels of IL-21R expression, suggesting a potential role for CD4 help in regulating the differentiation of this subset. Strikingly, depletion of CD4 T-cells or deletion of IL-21R signaling in P14 transgenic CD8 T-cells abrogated the development of the CX3CR1 CD8 subset, indicating a critical role for CD4 help in facilitating the differentiation of exhausted CD8 T-cells into a potent cytotoxic CD8 subset. Collectively, our work supports a new model of CD8 T-cell differentiation during chronic viral infection and has important implications for T-cell-based immunotherapies aimed at treating persistent infections and/or cancer. Citation Format: Ryan Zander, David Schauder, Gang Xin, Christine Nguyen, Xiaopeng Wu, Weiguo Cui. Single-cell RNA-sequencing (ScRNA-seq) reveals broad heterogeneity among CD8 T-cells during chronic viral infection and identifies a critical role for CD4 help in promoting the differentiation of a potent cytotoxic CD8 T-cell subset [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B200.