Faculty Bibliography

Programmed cell death protein 1 (PD-1) ligation delimits immunogenic responses in T cells. However, the consequences of programmed cell death 1 ligand 1 (PD-L1) ligation in T cells are uncertain. We found that T cell expression of PD-L1 in cancer was regulated by tumor antigen and sterile inflammatory cues. PD-L1⁺ T cells exerted tumor-promoting tolerance via three distinct mechanisms: (1) binding of PD-L1 induced STAT3-dependent 'back-signaling' in CD4⁺ T cells, which prevented activation, reduced T_H1-polarization and directed T_H17-differentiation. PD-L1 signaling also induced an anergic T-bet^-IFN-γ^- phenotype in CD8⁺ T cells and was equally suppressive compared to PD-1 signaling; (2) PD-L1⁺ T cells restrained effector T cells via the canonical PD-L1-PD-1 axis and were sufficient to accelerate tumorigenesis, even in the absence of endogenous PD-L1; (3) PD-L1⁺ T cells engaged PD-1⁺ macrophages, inducing an alternative M2-like program, which had crippling effects on adaptive antitumor immunity. Collectively, we demonstrate that PD-L1⁺ T cells have diverse tolerogenic effects on tumor immunity.

An increasing number of technologies combine spatial discrimination with molecule identification, as has been done for decades through immunohistochemistry and in situ hybridization. The difference is that now a sleuth of commercially available platforms promises to significantly increase the number of targets that can be identified on a single sample. They also provide higher throughput in terms of number of samples that can be interrogated, and some offer semiautomation with straightforward sample preparation. All these characteristics set up the new spatial profiling technologies as attractive candidates for biomedical research facilities. However, to be able to discriminate between them it is necessary to share real world experiences using them in our cores. This panel session brings together several researchers with hands on experience using two or more spatial profiling technologies. These will include antibody based (Opal/Vectra Polaris, Ultivue, MIBI, Hyperion) and nucleic acid (Visium, GeoMx) based platforms. Each panelist will give a quick overview of the technology, its biochemical principles, costs, advantages and disadvantages as they have identified them and compared them to each other or a gold standard, if available. At the end, we will have an open discussion where the audience is encouraged to ask questions and provide their experiences

Identification of biomarkers is a major goal of personalized medicine. Large transcriptome screens have identified new targets and molecular signatures for disease sub-types. However, tissue spatial information, which fundamentally alters in vivo cell behavior and gene expression, is lost. To understand spatial context and validate bulk tissue screens, most researchers rely exclusively on antibodies and immunostaining assays. Unfortunately, this is either not the appropriate choice for some targets, such as long non-coding RNAs, or it is not feasible because no reliable antibodies exist. To address these issues, we have established an alternative work-flow incorporating RNA in situ hybridization (CISH and FISH), whole slide and/or multispectral scanning and image analysis. Methods: RNAscope technology; Leica SCN scanner or Vectra3 multispectral imaging system for image acquisition; ImageJ2/FIJI, R, InForm and Visiopharm software platforms for quantitative analysis. Results: Several laboratories have used this workflow to address their specific questions. For example, we established and validated RNAscope assays for signaling factor transcripts, which are now integrated into an ongoing clinical trial. In this case, all tested commercial antibodies failed the validation assay. We also assessed expression of LNC RNAs in prostate cancer and put in place protocols for normalizing probe quantification across samples. The analysis revealed that storage and/or sample preparation affected the detection of certain LNC RNAs more than others identifying important factors regarding banking specimens. Spatial heat map visualization of RNAscope probes revealed an unexpected distribution of inflammatory cytokine targets in the kidney which are now being further investigated. In conclusion, RNA ISH is a powerful alternative strategy for assessing the spatial distribution of specific cell populations and critical biomarkers within intact tissues. This approach coupled with sophisticated imaging modalities and downstream analysis support provides new collaborative opportunities for Core laboratories.

Several modalities of multiplex immunofluorescence histology currently available require significant time and resources to implement. Many research laboratories develop questions benefiting from multiplex staining and analysis but do not have the human resources and/or equipment to perform the assay. Furthermore, pilot studies use similar metrics to evaluate multiplex histology data making them ideal for a core laboratory setup. The objective of this study was to establish a semi-automated workflow for multiplex immunofluorescence staining and initial quantification of cell populations in whole slide microscopy scans. The requirements for the workflow included: A. minimal transfer of decision making from the researcher to core personnel (Semi-Automation), B. modifiable in terms of antigen targets and tissue types with minimal disruption to the process and C. reproducible across samples submitted at different time periods (eg patient samples).

Background/Purpose : T and B cells come together in ectopic lymphoid aggregates in myositis, suggesting that local T:B cell interactions could play a role in disease. T follicular helper cells are increased in circulation in patients with active myositis. We studied circulating Tfh cells from myositis patients using single-cell RNA-sequencing and examined the proximity of Tfh cells to B cells in patient biopsies. Methods : Tfh cells [CD3 + CXCR5 + PD-1 + CXCR3 neg and CD3 + CXCR5 + PD-1 + CXCR3 pos ] cells were sorted from peripheral blood and subsets were identified by chemokine markers to designate Tfh1 and Tfh2/17 cell subsets. RNA sequencing was performed on individual cells of (3 controls and 3 myositis patients) using Fluidigm C1 HT platform, and data were analyzed using a pseudo-temporal ordering using Monocle. Biopsies were stained using the OPAL standardized sequential immunofluorescence method for PD-1, CXCR5, CD19, and CD4 in human muscle, and machine learning was used to map proximity of B cells to all T-cells compared with Tfh cells. Results : We found various subsets within the Tfh pool, corresponding to Tfh1 and Tfh2/17 cells and some cells that looked to be transitioning between states. Tfh2/17 were enriched in myositis patients vs. controls. The Tfh2/17 cells demonstrated a type I interferon signature, while the Tfh1 cells had a type II interferon and proteasome signature. In tissue, we demonstrate Tfh cells in close proximity to B cells in lymphoid aggregates. Conclusion : Tfh cells are present in myositis biopsies juxtaposed to B cells, suggesting productive T:B interactions in the tissue. Tfh subsets in blood from patients demonstrate distinct pathological signatures when compared to controls

As with most cancer types, there remains a subset of B-cell acute lymphoblastic leukaemia (B-ALL) patients who will relapse and succumb to therapy-resistant disease. It is believed that tumour heterogeneity underpins therapy failure leading to a Darwinian model of clonal evolution, however, such studies do not account for the role of the bone marrow microenvironment in supporting leukaemia survival, progression and escape from treatment. Here, we perform single-cell RNA-Sequencing (scRNA-Seq) to generate a comprehensive map of the primary human B-ALL bone marrow immune microenvironment throughout three distinct stages of the human leukemic disease process: diagnosis, remission and relapse. These studies show extensive re-modelling of the immune microenvironment composition and cell-to-cell interactions throughout the course conventional chemotherapy, and uncover a role for inflammatory leukaemia-associated monocytes in promoting B-ALL pathogenesis in vivo. These monocytic subsets are predictive of Ph+ B-ALL patient event-free survival and when targeted in B-ALL animal models, lead to prolonged disease remission. Our profiling of the human B-ALL bone marrow immune microenvironment provides a greater understanding of the potential extrinsic regulators of B-ALL survival and may highlight previously unknown environmental factors influencing immune-based treatment approaches to high-risk B-ALL.

Imiquimod is a topical toll-like-receptor-7 agonist currently used for treating basal cell carcinoma. Recently, imiquimod has demonstrated tumor regression in melanoma and breast cancer skin metastases. However, the molecular perturbations induced by imiquimod in breast cancer metastases have not been previously characterized. Here, we describe transcriptomic profiles associated with responsiveness to imiquimod in breast cancer skin metastases. Baseline and post-treatment tumor samples from patients treated with imiquimod in a clinical trial were profiled using Nanostring technology. Through an integrative analytic pipeline, we showed that tumors from patients who achieved a durable clinical response displayed a permissive microenvironment, substantiated by the upregulation of transcripts encoding for molecules involved in leukocyte adhesion and migration, cytotoxic functions, and antigen presentation. In responding patients, Imiquimod triggered a strong T-helper-1 (Th-1)/cytotoxic immune response, characterized by the coordinated upregulation of Th-1 chemokines, migration of Th-1 and cytotoxic T cells into the tumor, and activation of immune-effector functions, ultimately mediating tumor destruction. In conclusion, we have shown that topical imiquimod can induce a robust immune response in breast cancer metastases, and this response is more likely to occur in tumors with a pre-activated microenvironment. In this setting, imiquimod could be utilized in combination with other targeted immunotherapies to increase therapeutic efficacy.

Sonic Hedgehog (SHH) signaling, a developmental pathway promoting lung mesenchymal expansion and differentiation during embryogenesis, has been increasingly recognized as a profibrotic factor in mature lung, where it might contribute to the pathogenesis of lung fibrosis. Pathway inhibition at the level of the downstream Gli transcription factors Gli1 and Gli2 (by GANT61) ameliorates lung fibrosis in the bleomycin model, whereas inhibition proximally at the level of HH ligand (by anti Hh antibody 5E1) or Smo (by GDC-0449) of the canonical pathway does not, implicating Gli1 and/or Gli2 as a key target. The fact that both the Gli1-labelled cell lineage and Gli1 expressing cells expand during fibrosis formation and contribute significantly to the pool of myofibroblasts in the fibrosis scars suggests a fibrogenic role for Gli1. Therefore to further dissect the roles of Gli1 and Gli2 in lung fibrosis we evaluated Gli1 KO and control mice in the bleomycin model. Monitoring of Gli1^+/+ (n = 12), Gli1^lZ/+ (n = 37) and Gli1^lZ/lZ (n = 18) mice did not reveal differences in weight loss or survival. Lung evaluation at the 21-day endpoint did not show differences in lung fibrosis formation (as judged by morphology and trichrome staining), Ashcroft score, lung collagen content, lung weight, BAL protein content or BAL cell differential count. Our data suggest that Gli1 is not required for bleomycin-induced lung fibrosis.

The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytes and lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB), are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstrate that LukED and HlgAB cause vascular congestion and derangements in vascular fluid distribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines (DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the critical target for lethality. Consistent with this, LukED and HlgAB injure primary human endothelial cells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells are resistant to toxin-mediated lethality. During bloodstream infection in mice, DARC targeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potential for S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulence factors.