Search

Immune checkpoint blockade (ICB) therapy has emerged as viable first-line treatment for cancers such as melanoma. Despite the striking long-term clinical benefits that were previously unattainable, the effectiveness of ICB therapy remains limited in a majority of patients and cancer types. This is due in part to insufficient activation and/or restoration of anti-tumor T cell responses. Thus, understanding the signaling pathways that potentiate T cell exhaustion is essential for developing pharmacologic drugs that reactivate T cells and overcome the limitations of current ICB therapy. To this end, we developed a biologically relevant phenotypic screening system using the LCMV-CL13 infection model coupled with high-throughput flow cytometry to identify small-molecules that resurrect hypofunctional T cells. We discovered 19 hits following a screen of a manually curated collection of 12,000 repurposed drugs. Among the lead compounds identified were janus kinase (JAK) inhibitors including the anti-myelofibrosis drug ruxolitinib. Phenotypic rescue of exhausted T cells in vitro following ruxolitinib treatment resulted in a unique transcriptional signature that diverged from anti-PD-L1 treatment. Lineage-defining genes known to preserve precursor exhausted CD8+ T cells, including transcription factor 7 (Tcf7), were significantly upregulated following exposure to ruxolitinib. Mechanistically, ruxolitinib attenuated the cumulative pSTAT signaling signature within resurrected cells to enhance their survival. Collectively, these results demonstrate a disease-relevant framework for identifying small-molecule modulators of dysfunctional T cells and suggest that JAKs are viable targets for cancer immunotherapy.

ISG15 is one of the strongest type I interferon (IFN-I) stimulated genes (ISGs) which is conjugated to proteins in an enzymatic process termed ISGylation. Protein ISGylation is subsequently reversed by the enzyme Usp18 which acts in a de-ubiqutinase fashion to remove SIG15 from target proteins. Many proteins are ISGylated following IFN-I signaling however, immunological studies have been limited to the anti-viral properties of ISG15 conjugation while its regulation of innate and adaptive immune responses has been poorly studied. Here using Usp18−/− and enzymatically inactive (Usp18C61A/C61A Knock-in) mouse models, we show that hyper-ISGylation during chronic viral infection leads to severe immune pathology characterized by hematological disruptions, cytokine amplification, lung vascular leakage and death. The pathology associated with chronic infection required T cells but not T cell-intrinsic Usp18 expression. We reveal that lack of Usp18 in LysM+ cells reproduced the pathological manifestations observed in Usp18−/− or Usp18C61A/C61A mice. Using single cell sequencing, we observed that hyper-ISGylation during chronic infection altered neutrophil maturation and promoted an inflammatory neutrophil lung infiltrate. Importantly, neutrophil depletion reversed morbidity and mortality in Usp18C61A/C61A mice. In summary, we reveal Usp18’s enzymatic function is crucial for regulating inflammatory neutrophil differentiation and preventing immune hyperactivation during chronic viral infection. Moreover, our results suggest that hyper-ISGylation may drive the inflammatory pathology observed in Usp18-null patients and indicate that regulation of ISG15 expression or conjugation could lessen disease.

Emerging evidence indicates that B lymphocytes regulate immunity by producing antibodies, presenting antigens and secreting cytokines that provide signals to other cells to modulate both humoral and cellular immunity. Here, we show that B cells produce IL-27 upon activation by TLR4 and CD40 in the presence of IL-21 in vitro. Using Lymphocytic choriomeningitis virus (LCMV) Clone 13 as a model of a persistent viral infection, we identify plasma B cells as major source of IL-27. Further, we show that IL-27-producing B cells are key players in the regulation of immunity. Mice with conditional B cell–specific IL-27p28 knockout were unable to control persistent LCMV infection and failed to respond to anti-IFNAR1 immunotherapy. The impaired immunity found in these mice was associated with reduced virus-specific IgG2a/2c. In addition, these mice displayed reduced virus-specific CD4+ T cells and T follicular helper (Tfh) cells, suggesting that B-cell-derived cytokines regulate immunity by providing signals to T cells. The effects of Il27ra deletion from T cells mirrors the phenotypes observed in mice lacking B-cell-derived IL-27. In addition, IL-27ra deficient (KO) Tfh cells lost their ability to produce IL-21, resulting in aberrant production of virus-specific antibodies to LCMV clone 13 in IL-27ra KO mice. Finally, RNA-sequencing of virus-specific CD4+ T cells identified IL-27R-dependent genes during persistent LCMV infection. Collectively, our data reveal a central role of plasma cell-derived IL-27 in the regulation of T cell functions. Importantly, our study describes cellular interactions between T and B cells and highlights the importance of virus-specific IgG2a/2c during persistent LCMV infection.

B cells regulate immune responses by both generating antibodies and through cell surface interactions and cytokine production that provide signals to various cells to modulate humoral and cell-mediated immunity. During persistent viral infection, IL-27R is required for viral control which correlates with impaired anti-viral CD4+ T cell responses and lower LCMV-specific antibody production. Here, using mice with conditional B cell–specific IL-27 p28 deletion, we identify IL-27-producing B cells as key players in promoting control of persistent viral infection. Mice in which B cells lacked IL-27 p28 expression lost the ability to control persistent LCMV infection. The impaired immunity was associated with the reduction of GC B cells, plasma cells and Tfh cells. In addition, these mice also displayed reduced virus-specific CD8 T cells and lost the ability to expand CXCR5+ follicular CD8 T cells following anti-IFNAR1 treatment, suggesting that B-cell-derived IL-27 p28 regulates immunity by providing signals to T cells. To assess the role of IL-27 signaling in T cells we generated IL-27R conditional KO mice by mating IL-27Ra-floxed mice with CD4 and Granzyme B-Cre strains. Mice in which IL-27Ra was conditionally ablated in all T cells lost the ability to control persistent LCMV infection. In addition, these mice produced significantly lower titers of virus-specific IgG2a throughout the course of infection despite similar levels of virus-specific IgG1. Finally, we identify plasma cells expressing the transcription factor Blimp1 as a major source of IL-27 p28 during persistent LCMV infection. Overall, our study highlights the importance of IL-27-producing B cells in regulating T cell responses and control of persistent viral infection.