Neoadjuvant in situ vaccination with cowpea mosaic virus as a novel therapy against canine inflammatory mammary cancer.

BACKGROUND: Inflammatory mammary cancer (IMC), the counterpart of human inflammatory breast cancer (IBC), is the deadliest form of canine mammary tumors. IMC patients lack specific therapy and have poor outcomes. This proof-of-principle preclinical study evaluated the efficacy, safety, and effect on survival of neoadjuvant intratumoral (in situ) empty cowpea mosaic virus (eCPMV) immunotherapy in companion dogs diagnosed with IMC. METHODS: Ten IMC-bearing dogs were enrolled in the study. Five dogs received medical therapy, and five received weekly neoadjuvant in situ eCPMV immunotherapy (0.2-0.4 mg per injection) and medical therapy after the second eCPMV injection. Efficacy was evaluated by reduction of tumor growth; safety by hematological and biochemistry changes in blood and plasma; and patient outcome by survival analysis. eCPMV-induced immune changes in blood cells were analyzed by flow cytometry; changes in the tumor microenvironment were evaluated by CD3 (T lymphocytes), CD20 (B lymphocytes), FoxP3 (Treg lymphocytes), myeloperoxidase (MPO; neutrophils), Ki-67 (proliferation index, PI; tumor cell proliferation), and Cleaved Caspase-3 (CC-3; apoptosis) immunohistochemistry. RESULTS: Two neoadjuvant in situ eCPMV injections resulted in tumor shrinkage in all patients by day 14 without systemic adverse events. Although surgery for IMC is generally not an option, reduction in tumor size allowed surgery in two IMC patients. In peripheral blood, in situ eCPMV immunotherapy was associated with a significant decrease of Treg+/CD8+ ratio and changes in CD8+Granzyme B+ T cells, which behave as a lagging predictive biomarker. In the TME, higher neutrophilic infiltration and MPO expression, lower tumor Ki-67 PI, increase in CD3+ lymphocytes, decrease in FoxP3+/CD3+ ratio (p<0.04 for all comparisons), and no changes in CC-3+ immunostainings were observed in post-treatment tumor tissues when compared with pretreatment tumor samples. eCPMV-treated IMC patients had a statistically significant (p=0.033) improved overall survival than patients treated with medical therapy. CONCLUSIONS: Neoadjuvant in situ eCPMV immunotherapy demonstrated anti-tumor efficacy and improved survival in IMC patients without systemic adverse effects. eCPMV-induced changes in immune cells point to neutrophils as a driver of immune response. Neoadjuvant in situ eCPMV immunotherapy could be a groundbreaking immunotherapy for canine IMC and a potential future immunotherapy for human IBC patients.

Attenuation of peripheral serotonin inhibits tumor growth and enhances immune checkpoint blockade therapy in murine tumor models.

Platelet-derived peripheral serotonin has pleiotropic effects on coagulation, metabolism, tissue regeneration, and cancer growth; however, the effect of serotonin on the tumor microenvironment remains understudied. Peripheral serotonin­deficient (Tph1−/−) mice displayed reduced growth of subcutaneous and orthotopically injected syngeneic murine pancreatic and colorectal cancers with enhanced accumulation of functional CD8+ T cells compared to control C57BL/6 mice, resulting in extended overall survival. Subcutaneous and orthotopic syngeneic tumors from Tph1−/− mice expressed less programmed cell death 1 ligand 1 (PD-L1), suggesting serotonin-mediated regulation. Serotonin enhanced expression of PD-L1 on mouse and human cancer cells in vitro via serotonylation, which is the formation of covalent bonds between glutamine residues and serotonin, resulting in activation of small G proteins. Serotonin concentrations in metastases of patients with abdominal tumors negatively correlated to the number of CD8+ tumor-infiltrating T cells. Depletion of serotonin cargo or inhibition of serotonin release from thrombocytes decreased growth of syngeneic pancreatic and colorectal tumors in wild-type mice, increased CD8+ T cell influx, and decreased PD-L1 expression. Pharmacological serotonin depletion with oral fluoxetine or intraperitoneal injection of the TPH1 inhibitor telotristat augmented the effects of programmed cell death protein 1 (PD-1) checkpoint blockade and triggered long-term tumor control in mice subcutaneously inoculated with syngeneic colorectal and pancreatic tumors. Overall, peripheral serotonin weakens effector functions of CD8+ T cells within tumors. Clinically approved serotonin targeting agents alone or in combination with PD-1 blockade provided long-term control of established tumors in murine models, warranting further investigation of the clinical translatability of these findings.

Molecular mechanisms and treatment modalities in equine Culicoides hypersensitivity.

Equine Culicoides hypersensitivity (CH) is the most common allergic condition in horses affecting the skin. This review focuses on immunopathology and molecular mechanisms of equine CH. The role of eosinophils is emphasized, as well as disease severity and the influence of long-term chronic allergen exposure on T helper (Th) 2 cells. Using current knowledge from human allergic disorders, similar effects are hypothesized in equine patients. Key aspects of CH diagnosis and treatment are discussed, focusing on allergen specific immunotherapy and allergen-independent approaches, such as targeting hypereosinophilia through interleukin-5 and allergic non-histaminic pruritus though interleukin-31.

A ribonucleoprotein transfection strategy for CRISPR/Cas9-mediated gene editing and single cell cloning in rainbow trout cells.

BACKGROUND: The advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology marked the beginning of a new era in the field of molecular biology, allowing the efficient and precise creation of targeted mutations in the genome of every living cell. Since its discovery, different gene editing approaches based on the CRISPR/Cas9 technology have been widely established in mammalian cell lines, while limited knowledge is available on genetic manipulation in fish cell lines. In this work, we developed a strategy to CRISPR/Cas9 gene edit rainbow trout (Oncorhynchus mykiss) cell lines and to generate single cell clone-derived knock-out cell lines, focusing on the phase I biotransformation enzyme encoding gene, cyp1a1, and on the intestinal cell line, RTgutGC, as example. RESULTS: Ribonucleoprotein (RNP) complexes, consisting of the Cas9 protein and a fluorescently labeled crRNA/tracrRNA duplex targeting the cyp1a1 gene, were delivered via electroporation. A T7 endonuclease I (T7EI) assay was performed on flow cytometry enriched transfected cells in order to detect CRISPR-mediated targeted mutations in the cyp1a1 locus, revealing an overall gene editing efficiency of 39%. Sanger sequencing coupled with bioinformatic analysis led to the detection of multiple insertions and deletions of variable lengths in the cyp1a1 region directed by CRISPR/Cas9 machinery. Clonal isolation based on the use of cloning cylinders was applied, allowing to overcome the genetic heterogeneity created by the CRISPR/Cas9 gene editing. Using this method, two monoclonal CRISPR edited rainbow trout cell lines were established for the first time. Sequencing analysis of the mutant clones confirmed the disruption of the cyp1a1 gene open reading frame through the insertion of 101 or 1 base pair, respectively. CONCLUSIONS: The designed RNP-based CRISPR/Cas9 approach, starting from overcoming limitations of transfection to achieving a clonal cell line, sets the stage for exploiting permanent gene editing in rainbow trout, and potentially other fish cells, for unprecedented exploration of gene function.

Cross-Reactivity and Functionality of Approved Human Immune Checkpoint Blockers in Dogs.

BACKGROUND: Rodent cancer models have limitations in predicting efficacy, tolerability and accompanying biomarkers of ICIs in humans. Companion dogs suffering from neoplastic diseases have gained attention as a highly relevant translational disease model. Despite successful reports of PD-1/PD-L1 blockade in dogs, no compounds are available for veterinary medicine. METHODS: Here, we assessed suitability of seven FDA-approved human ICIs to target CTLA-4 or PD-1/PD-L1 in dogs. Cross-reactivity and blocking potential was assessed using ELISA and flow cytometry. Functional responses were assessed on peripheral blood mononuclear cells (PBMCs) derived from healthy donors (n = 12) and cancer patient dogs (n = 27) as cytokine production after stimulation. Immune composition and target expression of healthy donors and cancer patients was assessed via flow cytometry. RESULTS: Four candidates showed cross-reactivity and two blocked the interaction of canine PD-1 and PD-L1. Of those, only atezolizumab significantly increased cytokine production of healthy and patient derived PBMCs in vitro. Especially lymphoma patient PBMCs responded with increased cytokine production. In other types of cancer, response to atezolizumab appeared to correlate with a lower frequency of CD8 T cells. CONCLUSIONS: Cross-functionality of atezolizumab encourages reverse translational efforts using (combination) immunotherapies in companion dog tumor patients to benefit both veterinary and human medicine.

Enzymatic Dissociation Induces Transcriptional and Proteotype Bias in Brain Cell Populations.

Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37 °C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4 °C. These findings emphasize the risk of introducing technical biases and biological artifacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.

OMIP-065: Dog Immunophenotyping and T-Cell Activity Evaluation with a 14-Color Panel.

Companion dogs are increasingly recognized as large-animal models of diseases such as cancer, infectious-, inflammatory-, or autoimmune diseases. At the same time, compared to human clinics, veterinarians have only a fraction of the treatment options available. To study the immunological aspects of canine diseases and ultimately develop or adapt human treatments for the dog, the methodology also needs to be in place. Such tools include robust and reliable flow cytometric panels. The purpose of the panel described here is to assess the immune cell composition and their functionality in the peripheral blood mononuclear cells (PBMCs) of dogs. Moreover, its "plug and play" composition allows for an in-depth analysis of T-cell responses in ex vivo assays (Table 1). Initially, this panel has been designed for the analysis of cryopreserved PBMCs to allow batched analysis and to reduce interexperimental variation. Withers and colleagues published a comparable and-to our knowledge-currently the most extensive canine panel to date (1). While their study focused on the aging and activation status of T cells in dogs, our panel is designed to look at a broader range of cells with a higher number of markers. This allows a more in-depth analysis of functional extracellular and intracellular markers. In addition, all antibodies in our proposed panel are directly labeled. In combination with suitable lymphocyte isolation protocols, this panel could potentially also be adapted to analyze tissue biopsies from various different organs. © 2020 International Society for Advancement of Cytometry.

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery.

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 µg of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

Neurons under T Cell Attack Coordinate Phagocyte-Mediated Synaptic Stripping.

Inflammatory disorders of the CNS are frequently accompanied by synaptic loss, which is thought to involve phagocytic microglia and complement components. However, the mechanisms accounting for aberrant synaptic connectivity in the context of CD8+ T cell-driven neuronal damage are poorly understood. Here, we profiled the neuronal translatome in a murine model of encephalitis caused by CD8+ T cells targeting antigenic neurons. Neuronal STAT1 signaling and downstream CCL2 expression were essential for apposition of phagocytes, ensuing synaptic loss and neurological disease. Analogous observations were made in the brains of Rasmussen's encephalitis patients. In this devastating CD8+ T cell-driven autoimmune disease, neuronal STAT1 phosphorylation and CCL2 expression co-clustered with infiltrating CD8+ T cells as well as phagocytes. Taken together, our findings uncover an active role of neurons in coordinating phagocyte-mediated synaptic loss and highlight neuronal STAT1 and CCL2 as critical steps in this process that are amenable to pharmacological interventions.

Rationale for Combining Bispecific T Cell Activating Antibodies With Checkpoint Blockade for Cancer Therapy.

T cells have been established as core effectors for cancer therapy; this has moved the focus of therapeutic endeavors to effectively enhance or restore T cell tumoricidal activity rather than directly target cancer cells. Both antibodies targeting the checkpoint inhibitory molecules programmed death receptor 1 (PD1), PD-ligand 1 (PD-L1) and cytotoxic lymphocyte activated antigen 4 (CTLA4), as well as bispecific antibodies targeting CD3 and CD19 are now part of the standard of care. In particular, antibodies to checkpoint molecules have gained broad approval in a number of solid tumor indications, such as melanoma or non-small cell lung cancer based on their unparalleled efficacy. In contrast, the efficacy of bispecific antibody-derivatives is much more limited and evidence is emerging that their activity is regulated through diverse checkpoint molecules. In either case, both types of compounds have their limitations and most patients will not benefit from them in the long run. A major aspect under investigation is the lack of baseline antigen-specific T cells in certain patient groups, which is thought to render responses to checkpoint inhibition less likely. On the other hand, bispecific antibodies are also restricted by induced T cell anergy. Based on these considerations, combination of bispecific antibody mediated on-target T cell activation and reversal of anergy bears high promise. Here, we will review current evidence for such combinatorial approaches, as well as ongoing clinical investigations in this area. We will also discuss potential evidence-driven future avenues for testing.

IL-12 protects from psoriasiform skin inflammation.

Neutralization of the common p40-subunit of IL-12/23 in psoriasis patients has led to a breakthrough in the management of moderate to severe disease. Aside from neutralizing IL-23, which is thought to be responsible for the curative effect, anti-p40 therapy also interferes with IL-12 signalling and type 1 immunity. Here we dissect the individual contribution of these two cytokines to the formation of psoriatic lesions and understand the effect of therapeutic co-targeting of IL-12 and IL-23 in psoriasis. Using a preclinical model for psoriatic plaque formation we show that IL-12, in contrast to IL-23, has a regulatory function by restraining the invasion of an IL-17-committed γδT (γδT17) cell subset. We discover that IL-12 receptor signalling in keratinocytes initiates a protective transcriptional programme that limits skin inflammation, suggesting that collateral targeting of IL-12 by anti-p40 monoclonal antibodies is counterproductive in the therapy of psoriasis.

Dermal IL-17-producing γδ T cells establish long-lived memory in the skin.

Conventional αß T cells have the ability to form a long-lasting resident memory T-cell (TRM ) population in nonlymphoid tissues after encountering foreign antigen. Conversely, the concept of 'innate memory', where the ability of nonadaptive branches of the immune system to deliver a rapid, strengthened immune response upon reinfection or rechallenge, is just emerging. Using the αß T-cell-independent Aldara psoriasis mouse model in combination with genetic fate-mapping and reporter systems, we identified a subset of γδ T cells in mice that is capable of establishing a long-lived memory population in the skin. IL-17A/F-producing Vγ4(+) Vδ4(+) T cells populate and persist in the dermis for long periods of time after initial stimulation with Aldara. Experienced Vγ4(+) Vδ4(+) cells show enhanced effector functions and mediate an exacerbated secondary inflammatory response. In addition to identifying a unique feature of γδ T cells during inflammation, our results have direct relevance to the human disease as this quasi-innate memory provides a mechanistic insight into relapses and chronification of psoriasis.

Langerin(neg) conventional dendritic cells produce IL-23 to drive psoriatic plaque formation in mice.

Psoriasis is an autoinflammatory skin disease of unknown etiology. Topical application of Aldara cream containing the Toll-like receptor (TLR)7 agonist Imiquimod (IMQ) onto patients induces flares of psoriasis. Likewise, in mice IMQ triggers pathological changes closely resembling psoriatic plaque formation. Key cytokines like IL-23 and type-I IFN (IFN-I), both being produced mainly by dendritic cells (DCs), have been implicated in psoriasis. Although plasmacytoid DCs (pDCs) are the main source of IFNα and thought to initiate disease, conventional DCs (cDCs) appear to maintain the psoriatic lesions. Any role of cDCs during lesion formation remains elusive. Here, we report that selective activation of TLR7 signaling specifically in CD11c(+) DCs was sufficient to induce psoriasiform skin disease in mice. Intriguingly, both pDCs and the IFN-I pathway were dispensable for the development of local skin inflammation. Selective TLR7 triggering of Langerin(+) DCs resulted in attenuated disease, whereas their depletion did not alter the severity of skin lesions. Moreover, after IMQ-painting, IL-23 was exclusively produced by Langerin(neg) DCs in vivo. In conclusion, TLR7-activated Langerin(neg) cDCs trigger psoriatic plaque formation via IL-23-mediated activation of innate IL-17/IL-22-producing lymphocytes, independently of pDCs or IFN-I. These results suggest therapeutic targeting of IL-23 production by cDCs to refine current treatment strategies for psoriasis.

Rorγt+ innate lymphocytes and γδ T cells initiate psoriasiform plaque formation in mice.

Psoriasis is a common, relapsing inflammatory skin disease characterized by erythematous scaly plaques. Histological manifestations of psoriasis include keratinocyte dysregulation and hyperproliferation, elongated rete ridges, and inflammatory infiltrates consisting of T cells, macrophages, dendritic cells, and neutrophils. Despite the availability of new effective drugs to treat psoriasis, the underlying mechanisms of pathogenesis are still poorly understood. Recent studies have shown that Aldara cream, used to treat benign skin abnormalities, triggers psoriasis-like disease in humans and mice and have implicated Th17 cells in disease initiation. Using this as a model, we found a predominant role for the Th17 signature cytokines IL-17A, IL-17F, and IL-22 in psoriasiform plaque formation in mice. Using gene-targeted mice, we observed that loss of Il17a, Il17f, or Il22 strongly reduced disease the severity of psoriasis. However, we found that Th17 cells were not the primary source of these pathogenic cytokines. Rather, IL-17A, IL-17F, and IL-22 were produced by a skin-invading population of γδ T cells and RORγt(+) innate lymphocytes. Furthermore, our findings establish that RORγt(+) innate lymphocytes and γδ T cells are necessary and sufficient for psoriatic plaque formation in an experimental disease model that closely resembles human psoriatic plaque formation.