A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues.

Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.

Multi-Parameter Quantitative Imaging of Tumor Microenvironments Reveals Perivascular Immune Niches Associated With Anti-Tumor Immunity.

Tumors are populated by a multitude of immune cell types with varied phenotypic and functional properties, which can either promote or inhibit anti-tumor responses. Appropriate localization and function of these cells within tumors is critical for protective immunity, with CD8 T cell infiltration being a biomarker of disease outcome and therapeutic efficacy. Recent multiplexed imaging approaches have revealed highly complex patterns of localization for these immune cell subsets and the generation of distinct tumor microenvironments (TMEs), which can vary among cancer types, individuals, and within individual tumors. While it is recognized that TMEs play a pivotal role in disease progression, a better understanding of their composition, organization, and heterogeneity, as well as how distinct TMEs are reshaped with immunotherapy, is necessary. Here, we performed spatial analysis using multi-parameter confocal imaging, histocytometry, and CytoMAP to study the microanatomical organization of immune cells in two widely used preclinical cancer models, the MC38 colorectal and KPC pancreatic murine tumors engineered to express human carcinoembryonic antigen (CEA). Immune responses were examined in either unperturbed tumors or after immunotherapy with a CEA T cell bispecific (CEA-TCB) surrogate antibody and anti-PD-L1 treatment. CEA-TCB mono and combination immunotherapy markedly enhanced intra-tumoral cellularity of CD8 T cells, dominantly driven by the expansion of TCF1-PD1+ effector T cells and with more minor increases in TCF1+PD1+ resource CD8 T cells. The majority of infiltrating T cells, particularly resource CD8 T cells, were colocalized with dendritic cells (DCs) or activated MHCII+ macrophages, but largely avoided the deeper tumor nest regions composed of cancer cells and non-activated macrophages. These myeloid cell - T cell aggregates were found in close proximity to tumor blood vessels, generating perivascular immune niches. This perivascular TME was present in untreated samples and markedly increased after CEA-TCB therapy, with its relative abundance positively associated with response to therapy. Together, these studies demonstrate the utility of advanced spatial analysis in cancer research by revealing that blood vessels are key organizational hubs of innate and adaptive immune cells within tumors, and suggesting the likely relevance of the perivascular immune TME in disease outcome.

CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment.

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.

Organotypic tumor slice cultures provide a versatile platform for immuno-oncology and drug discovery.

Organotypic tumor slices represent a physiologically-relevant culture system for studying the tumor microenvironment. Systematic characterization of the tumor slice culture system will enable its effective application for translational research. Here, using flow cytometry-based immunophenotyping, we performed a comprehensive characterization of the immune cell composition in organotypic tumor slices prepared from four syngeneic mouse tumor models and a human liver tumor. We found that the immune cell compositions of organotypic tumor slices prepared on the same day as the tumor cores were harvested are similar. Differences were primarily observed in the lymphocyte population of a clinical hepatocellular carcinoma case. Viable populations of immune cells persisted in the tumor slices for 7 days. Despite some changes in the immune cell populations, we showed the utility of mouse tumor slices for assessing responses to immune-modulatory agents. Further, we demonstrated the ability to use patient-derived xenograft tumor slices for assessing responses to targeted and cytotoxic drugs. Overall, tumor slices provide a broadly useful platform for studying the tumor microenvironment and evaluating the preclinical efficacy of cancer therapeutics.

Protective Role of Myeloid Cells Expressing a G-CSF Receptor Polymorphism in an Induced Model of Lupus.

The genetic analysis of the lupus-prone NZM2410 mouse has identified a suppressor locus, Sle2c2, which confers resistance to spontaneous lupus in combination with NZM2410 susceptibility loci, or in the chronic graft-versus-host disease (cGVHD) induced model of lupus in the B6.Sle2c2 congenic strain. The candidate gene for Sle2c2, the Csf3r gene encoding the granulocyte colony-stimulating factor receptor (G-CSF-R/CD114), was validated when cGVHD was restored in B6.Sle2c2 mice after treatment with G-CSF. The goal of the project reported herein was to investigate the myeloid cells that confer resistance to cGVHD and to ascertain if the mechanism behind their suppression involves the G-CSF pathway. We showed that despite expressing the highest levels of G-CSF-R, neutrophils play only a modest role in the autoimmune activation induced by cGVHD. We also found reduced expression levels of G-CSF-R on the surface of dendritic cells (DCs) and a differential distribution of DC subsets in response to cGVHD in B6.Sle2c2 versus B6 mice. The CD8α+ DC subset, known for its tolerogenic phenotype, was expanded upon induction of cGVHD in B6.Sle2c2 mice. In addition, the deficiency of CD8α+ DC subset enhanced the severity of cGVHD in B6.Batf3-/- and B6.Sle2c2 mice, confirming their role in suppression of cGVHD. B6.Sle2c2DCs presented lowered activation and antigen presentation abilities and expressed lower levels of genes associated with DC activation and maturation. Exposure to exogenous G-CSF reversed the majority of these phenotypes, suggesting that tolerogenic DCs maintained through a defective G-CSF-R pathway mediated the resistance to cGVHD in B6.Sle2c2 mice.

Immune Cell Metabolism in Systemic Lupus Erythematosus.

Cellular metabolism represents a newly identified checkpoint of effector functions in the immune system. A solid body of work has characterized the metabolic requirements of normal T cells during activation and differentiation into polarized effector subsets. Similar studies have been initiated to characterize the metabolic requirements for B cells and myeloid cells. Only a few studies though have characterized the metabolism of immune cells in the context of autoimmune diseases. Here, we review what is known on the altered metabolic patterns of CD4+ T cells, B cells, and myeloid cells in lupus patients and lupus-prone mice and how they contribute to lupus pathogenesis. We also discuss how defects in immune metabolism in lupus can be targeted therapeutically.

Metabolic Factors that Contribute to Lupus Pathogenesis.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which organ damage is mediated by pathogenic autoantibodies directed against nucleic acids and protein complexes. Studies in SLE patients and in mouse models of lupus have implicated virtually every cell type in the immune system in the induction or amplification of the autoimmune response as well as the promotion of an inflammatory environment that aggravates tissue injury. Here, we review the contribution of CD4+ T cells, B cells, and myeloid cells to lupus pathogenesis and then discuss alterations in the metabolism of these cells that may contribute to disease, given the recent advances in the field of immunometabolism.

The granulocyte colony stimulating factor pathway regulates autoantibody production in a murine induced model of systemic lupus erythematosus.

INTRODUCTION: An NZB-derived genetic locus (Sle2c2) that suppresses autoantibody production in a mouse model of induced systemic lupus erythematosus contains a polymorphism in the gene encoding the G-CSF receptor. This study was designed to test the hypothesis that the Sle2c2 suppression is associated with an impaired G-CSF receptor function that can be overcome by exogenous G-CSF. METHODS: Leukocytes from B6.Sle2c2 and B6 congenic mice, which carry a different allele of the G-CSF receptor, were compared for their responses to G-CSF. Autoantibody production was induced with the chronic graft-versus-host-disease (cGVHD) model by adoptive transfer of B6.bm12 splenocytes. Different treatment regimens varying the amount and frequency of G-CSF (Neulasta®) or carrier control were tested on cGVHD outcomes. Autoantibody production, immune cell activation, and reactive oxygen species (ROS) production were compared between the two strains with the various treatments. In addition, the effect of G-CSF treatment was examined on the production autoantibodies in the B6.Sle1.Sle2.Sle3 (B6.TC) spontaneous model of lupus. RESULTS: B6.Sle2c2 and B6 leukocytes responded differently to G-CSF. G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment. G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils. In contrast, the expression of G-CSF responsive genes indicated a higher G-CSF receptor signaling in B6.Sle2c2 cells. G-CSF treatment restored the ability of B6.Sle2c2 mice to produce autoantibodies in a dose-dependent manner upon cGVHD induction, which correlated with restored CD4+ T cells activation, as well as dendritic cell and granulocyte expansion. Steady-state ROS production was higher in B6.Sle2c2 than in B6 mice. cGVHD induction resulted in a larger increase in ROS production in B6 than in B6.Sle2c2 mice, and this difference was eliminated with G-CSF treatment. Finally, a low dose G-CSF treatment accelerated the production of anti-dsDNA IgG in young B6.TC mice. CONCLUSION: The different in vivo and in vitro responses of B6.Sle2c2 leukocytes are consistent with the mutation in the G-CSFR having functional consequences. The elimination of Sle2c2 suppression of autoantibody production by exogenous G-CSF indicates that Sle2c2 corresponds to a loss of function of G-CSF receptor. This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus. Overall, these results suggest that the G-CSF pathway regulates the production of autoantibodies in murine models of lupus.

Mapping the T helper cell response to acid α-glucosidase in Pompe mice.

Pompe disease is a neuromuscular disease caused by an inherited deficiency of the lysosomal enzyme acid α-glucosidase (GAA). The resulting accumulation of glycogen causes muscle weakness with the severe form of the disease resulting in death by cardiorespiratory failure in the first year of life. The only available treatment, enzyme replacement therapy (ERT) with recombinant GAA (rhGAA), is severely hampered by antibody responses that reduce efficacy and cause immunotoxicities. Currently, Pompe mice represent the only pre-clinical model for development of new treatments and for immunological studies. While antibody formation following ERT in this model has been described, the underlying T cell response has not been studied. In order to define the T helper response to rhGAA in Pompe mice, immunodominant CD4(+) T cell epitopes were mapped in GAA(-/-) 129SVE mice using ELISpot. Additionally, cytokine responses and antibody formation against rhGAA during ERT were measured. Among the three CD4(+) T cell epitopes identified, only epitope IFLGPEPKSVVQ, predicted to be the strongest MHC II binder, consistently contributed to IL-4 production. Frequencies of IL-4 producing T cells were considerably higher than those of IL-17 or IFN-γ producing cells, suggesting a predominantly Th2 cell mediated response. This is further supported by IgG1 being the prevalent antibody subclass against rhGAA during ERT and consistent with prior reports on IgE formation and anaphylaxis in this model. These results will facilitate mechanistic studies of the immune response to rhGAA in Pompe mice during development of new therapies and tolerance protocols.