<scp>OMIP‐065</scp>: Dog Immunophenotyping and T‐Cell Activity Evaluation with a 14‐Color Panel

Pantelyushin, Stanislav; Ranninger, Elisabeth; Bettschart‐Wolfensberger, Regula; Berg, Johannes vom

doi:10.1002/cyto.a.24168

Cited by 10 publications

(14 citation statements)

References 15 publications

(21 reference statements)

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“…To compare the atezolizumab responders (R) vs. non-responders (NR) PBMCs we characterized their immune subset composition using our recently developed optimized multicolor immunofluorescence panel [ 26 ]. We could clearly detect all major cell lineages in the cancer patient PBMCs, which matched the ones from healthy donors, including monocytes, neutrophils, B cells, NK cells, as well as CD4+, CD8+ or double negative T cells ( Figure 4 G,H).…”

Section: Resultsmentioning

confidence: 99%

“…Human ICIs were detected by flow cytometry using anti-human Fc PE labeled antibody (clone HP6017, 409304, Biolegend) at 1:200 dilution. Canine PBMC were analyzed with the panel previously described ( Table 1 ) [ 26 ]. Ki67 AF700 staining was performed using Foxp3/Transcription Factor Staining Buffer Set (00-5523-00, Thermo Fisher) according to manufacturer’s instructions (clone SolA15, 56-5698-82, Thermo Fisher).…”

Section: Methodsmentioning

confidence: 99%

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

Pantelyushin

Ranninger

Guerrera

et al. 2021

Cancers

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Pantelyushin

Ranninger

Guerrera

et al. 2021

Cancers

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“…Flow cytometry analysis was performed using a 14-color panel (online supplemental table S2) using reagents and procedures as previously described. 26 Stained samples were acquired using LSR Fortessa II (BD Biosciences, San Jose, California, USA) equipped with 4 lasers and 16 detectors. Flow cytometric analysis was performed using FlowJo software (V.10.7.1; BD Bioscience).…”

Section: Quality Of Life and Tumor Response Evaluationmentioning

confidence: 99%

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

Alonso-Miguel

Valdivia

Guerrera

et al. 2022

J Immunother Cancer

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BackgroundInflammatory 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.MethodsTen 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.ResultsTwo 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.ConclusionsNeoadjuvant 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.

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“…Based on reports in humans and dogs, markers for regulatory T cells (FoxP3 and CD25), proliferation/activation, (Ki67), (granzyme B), and functionality and T cell programming (IFN-γ) were feasible for a single panel and provided a diverse set of parameters for a preliminary assessment of T cell phenotyping of canine melanoma patients and controls. Limitations of the selected markers are acknowledged and additional T cell markers currently available would include eomes ( 34 ), PD-1 ( 25 , 91 , 92 ), T-bet and GATA-3 ( 44 ), memory markers CD62L and CD45RA ( 33 ) and additional other markers including cytokines including such as TNF-α.…”

Section: Discussionmentioning

confidence: 99%

“…However, additional canine-specific or cross-reactive reagents for critical immune markers are now emerging rapidly (25)(26)(27)(28)(29)(30)(31)(32). These new reagents and tools have encouraged the development of diagnostic and experimental tools (33)(34)(35)(36)(37). This will facilitate examination of the canine immune response to melanoma and immunologic interventions for multiple cancers including melanoma.…”

Section: Introductionmentioning

confidence: 99%

T Cell Immune Profiles of Blood and Tumor in Dogs Diagnosed With Malignant Melanoma

et al. 2021

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Investigation of canine T cell immunophenotypes in canine melanomas as prognostic biomarkers for disease progression or predictive biomarkers for targeted immunotherapeutics remains in preliminary stages. We aimed to examine T cell phenotypes and function in peripheral blood mononuclear cells (PBMC) and baseline tumor samples by flow cytometry, and to compare patient (n = 11–20) T cell phenotypes with healthy controls dogs (n = 10–20). CD3, CD4, CD8, CD25, FoxP3, Ki67, granzyme B, and interferon-γ (IFN-γ) were used to classify T cell subsets in resting and mitogen stimulated PBMCs. In a separate patient cohort (n = 11), T cells were classified using CD3, CD4, CD8, FoxP3, and granzyme B in paired PBMC and single cell suspensions of tumor samples. Analysis of flow cytometric data of individual T cell phenotypes in PBMC revealed specific T cell phenotypes including FoxP3+ and CD25+FoxP3- populations that distinguished patients from healthy controls. Frequencies of IFN-γ+ cells after ConA stimulation identified two different patient phenotypic responses, including a normal/exaggerated IFN-γ response and a lower response suggesting dysfunction. Principle component analysis of selected T cell immunophenotypes also distinguished patients and controls for T cell phenotype and revealed a clustering of patients based on metastasis detected at diagnosis. Findings supported the overall hypothesis that canine melanoma patients display a T cell immunophenotype profile that is unique from healthy pet dogs and will guide future studies designed with larger patient cohorts necessary to further characterize prognostic T cell immunophenotypes.

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

Cited by 10 publications

References 15 publications

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

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

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

T Cell Immune Profiles of Blood and Tumor in Dogs Diagnosed With Malignant Melanoma

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