Canine CD4 Research Articles

The CCL2-CCR4 axis promotes Regulatory T cell trafficking to canine glioma tissues

PurposeSpontaneously occurring glioma in pet dogs is increasingly recognized as a valuable translational model for human glioblastoma. Canine high-grade glioma and human glioblastomas share many molecular similarities, including the accumulation of immunosuppressive regulatory T cells (Tregs) that inhibit anti-tumor immune responses. Identifying in dog mechanisms responsible for Treg recruitment may afford to target the cellular population driving immunosuppression, the results providing a rationale for translational clinical studies in human patients. Our group has previously identified C-C motif chemokine 2 (CCL2) as a glioma-derived T-reg chemoattractant acting on chemokine receptor 4 (CCR4) in a murine orthotopic glioma model. Recently, we demonstrated a robust increase of CCL2 in the brain tissue of canine patients bearing high-grade glioma.MethodsWe performed a series of in vitro experiments using canine Tregs and patient-derived canine glioma cell lines (GSC 1110, GSC 0514, J3T-Bg, G06A) to interrogate the CCL2-CCR4 signaling axis in the canine.ResultsWe established a flow cytometry gating strategy for identifying and isolating FOXP3+ Tregs in dogs. The canine CD4 + CD25high T-cell population was highly enriched in FOXP3 and CCR4 expression, indicating they are bona fide Tregs. Canine Treg migration was enhanced by CCL2 or by glioma cell line-derived supernatant. Blockade of the CCL2-CCR4 axis significantly reduced migration of canine Tregs. CCL2 mRNA was expressed in all glioma cell lines, and expression increased when exposed to Tregs but not CD4 + helper T-cells.ConclusionOur study validates CCL2-CCR4 as a bi-directional Treg-glioma immunosuppressive and tumor-promoting axis in canine high-grade glioma.

The CCL2-CCR4 Axis Promotes Regulatory T Cell Trafficking to Canine Glioma Tissues.

Spontaneously occurring glioma in pet dogs is increasingly recognized as a valuable translational model for human glioblastoma. Canine high grade glioma and human glioblastomas share many molecular similarities, including accumulation of immunosuppressive regulatory T cells (Tregs) that inhibit anti-tumor immune responses. Identifying in dog mechanisms responsible for Treg recruitment may afford targeting the cellular population driving immunosuppression, the results providing a rationale for translational clinical studies in human patients. Our group has previously identified C-C motif chemokine 2 (CCL2) as a glioma-derived T-reg chemoattractant acting on chemokine receptor 4 (CCR4) in a murine orthotopic model of glioma. Recently, we demonstrated a robust increase of CCL2 in the brain tissue of canine patients bearing high-grade glioma. We performed a series of in vitro experiments using canine Tregs and patient-derived canine glioma cell lines (GSC 1110, GSC 0514, J3T-Bg, G06A) to interrogate the CCL2-CCR4 signaling axis in the canine. We established a flow cytometry gating strategy for identification and isolation of FOXP3+ Tregs in dogs. The canine CD4 + CD25high T-cell population was highly enriched in FOXP3 and CCR4 expression, indicating they are bona fide Tregs. Canine Treg migration was enhanced by CCL2 or by glioma cell line-derived supernatant. Blockade of the CCL2-CCR4 axis significantly reduced migration of canine Tregs. CCL2 mRNA was expressed in all glioma cell lines and expression increased when exposed to Tregs but not to CD4 + helper T-cells. Our study validates CCL2-CCR4 as a bi-directional Treg-glioma immunosuppressive and tumor-promoting axis in canine high-grade glioma.

A conserved TRAF binding motif within human 41BB enhances canine CD8+ CAR T cell expansion and cytolysis

Abstract Anti-CD19 chimeric antigen receptor T cells (CART) have transformed cancer therapy by inducing durable remissions of B cell cancers, although incomplete responses are common, highlighting a need for improved therapy. Immune competent dogs develop B cell lymphomas with similar pathophysiology as humans, offering a compelling opportunity for CART preclinical assessment. In a first-in-canine trial, anti-CD20 CART with canine 41BB and CD3ζ (caBBζ) domains exerted selective pressure on B cell lymphomas but did not persist or induce remission. We hypothesize canine CART signaling defects impair cytolytic activity and persistence. To optimize CAR design, we incorporated canine CD28 and CD3ζ (ca28ζ) or human (hu) BBζ and evaluated CART function and immunophenotype, using caBBζ as a reference control. In both an in vitro rechallenge and canine leukemia xenograft model, huBBζ CART elicited greater cytolysis and CD8 T cell outgrowth than caBBζ, and ca28ζ CART did not persist. Mutagenesis studies indicate T235 in the conserved PxQxT TRAF binding motif is required for CD8 outgrowth in huBBζ CART. Dogs and other mammals express A235, but introduction of A235T is not sufficient to confer CD8 outgrowth in caBBζ CART. Ongoing studies are profiling pathways downstream of ca/hu41BB that drive cytolytic activity and CD8 outgrowth. These studies reveal fundamental differences in T cell biology between humans and other mammals that hold promise for enabling clinical efficacy of canine CART.

Abstract 1772: A naturally occurring canine model of peripheral T-cell lymphoma, not otherwise specified

Abstract Despite being the most common subtype of human peripheral T-cell lymphoma (PTCL), PTCL-not otherwise specified (PTCL-NOS) remains a poorly understood diagnosis of exclusion with poor survival times and treatment responses. Canine PTCL clinically and immunophenotypically resembles human PTCL-NOS, and PTCL is more common in dogs than humans, leading to their interest as a naturally occurring preclinical model. Canine models of cancer offer several unique advantages: in contrast to traditional rodent models, these are spontaneous tumors in genetically diverse and immunocompetent animals living in a shared environment with humans, but relative to their human counterparts, they offer accelerated lifespans with shorter clinical courses of disease. Here, we analyzed the gene expression profile of 96 canine CD4+ PTCLs to determine similarities to human PTCL-NOS and investigate a possible cell of origin. Bulk RNA-seq was performed on lymph node aspirates of 96 cases of canine CD4+ PTCL diagnosed by flow cytometry and sorted CD4+ nodal lymphocytes and CD4+ thymocytes from healthy control dogs. Raw reads were aligned to the Ensembl canine reference genome (ROS_Cfam_1.0) and tabulated. Data normalization and differential expression analysis was conducted with DESeq2, and differentially expressed genes were compared to human PTCL-NOS and various stages of T-cell development via gene set enrichment analyses (GSEA). Global gene expression in canine CD4+ PTCL was analogous to that of human PTCL-NOS (NES = 2.3, p < 0.001). Additionally, canine CD4+ PTCL had increased expression of GATA3 (log2fc = 1.9, padj = 6.5e-07) and was enriched for gene signatures associated with downregulation of PTEN (NES = 1.7, p = 0.0001) and upregulation of PI3K/AKT/mTOR signaling (NES = 1.5, padj = 0.0007), resembling the more aggressive human GATA3-PTCL subtype. Canine CD4+ PTCL was also enriched for human and murine gene signatures associated with early thymocyte progenitor cells and had increased expression of genes of immaturity, including CD34 (log2fc = 7.8, padj = 2.6e-14), KIT (log2fc = 3.3, padj = 7.7e-05), and CCR9 (log2fc = 4.8, padj = 3.9e-11), although surface CD34 expression is not detected by flow cytometry. These findings were confirmed when we compared the canine PTCL gene expression programs to a canine single cell transcriptomic atlas of hematopoietic precursors and T cells across canine bone marrow, thymic, and lymph node tissues. Canine CD4+ PTCL cells were significantly (padj < 0.05) enriched for gene signatures associated with early thymic and bone marrow precursors (NES 1.3-2.1), and negatively enriched for gene signatures associated with naïve nodal CD4+ T cells (NES = -3.2, padj = 0.006). In conclusion, the gene expression profile of canine CD4+ PTCL resembles human PTCL-NOS, supporting its potential as a naturally occurring preclinical model, and a subset of these neoplasms may arise from a thymic precursor cell of origin. Citation Format: Eileen Owens, Adam Harris, Anne Avery. A naturally occurring canine model of peripheral T-cell lymphoma, not otherwise specified [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1772.

Abstract 2837: Conservation of JAK-STAT signaling in canine lymphocytes responding to human IL-6, IL-7, and IL-1 alpha

Abstract Dogs share physiological similarities with humans, positioning them as valuable models for studying the efficacy and safety of drugs and their underlying mechanisms of action. The JAK-STAT pathway, a highly conserved signaling pathway across species, including humans and dogs, presents an opportunity to investigate the therapeutic potential of cytokines that utilize this pathway. In this study, we aimed to evaluate the functional activity of three canine cytokines on canine cells. To assess stimulation of the JAK/STAT pathway, we used a flow cytometry assay that measures phospho-STAT in activated canine or human lymphocytes in whole blood specimens. Testing of commercially available human and canine IL-6 proteins revealed that canine lymphocytes respond poorly to most canine IL-6 products, whereas all recombinant human IL-6 products stimulated STAT phosphorylation in canine lymphocytes, despite sharing low sequence similarity with canine IL-6. In human T cells, both canine IL-6 and human IL-6 stimulated STAT phosphorylation, demonstrating biological activity from the canine IL-6 products. Several JAK inhibitors inhibited cytokine-induced STAT phosphorylation in both canine and human CD4 T-cells, consistent with JAK inhibition, but targeting the IL-6 receptor with tocilizumab only blocked IL-6-induced STAT phosphorylation in human but not canine CD4 T-cells in whole blood. These findings demonstrate the potential of our assay system to assess cross-species cytokine reactivity, determine if JAK/STAT inhibitors used clinically might be useful in treating inflammatory conditions in dogs, and identify novel canine-specific inhibitors. Additionally, we explored the induction of IL-8 by human and canine IL-1 alpha in IL-1 alpha sensitive human cells. Human IL-1 alpha failed to stimulate a canine IL-1 alpha sensitive cell line, suggesting species-specific differences in IL-1 alpha activity. Furthermore, we discovered that canine IL-7 and human IL-7 are species-specific. In summary, this proof-of-concept study highlights the capability to assess the activation of STATs and pathway inhibition by JAK inhibitors in canine whole blood using flow cytometry. We confirmed that human IL-6 stimulates JAK/STAT signaling in canine CD4 T-cells. Our results also revealed significant differences in species cross-reactivity of human IL-6, IL-7, and IL-1 alpha, underscoring the importance of species-specific investigations. Leveraging the similarities and differences between canine and human cytokines targeting the JAK-STAT pathway can enhance drug development efforts and contribute to the advancement of safer and more effective therapies for both humans and pet dogs with similar diseases. This study was funded by NCI Contract No. 75N91019D00024. Citation Format: King Leung Fung, Melissa L. Breiner, James B. Mitchell, Ralph E. Parchment. Conservation of JAK-STAT signaling in canine lymphocytes responding to human IL-6, IL-7, and IL-1 alpha [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2837.

The gene expression profile and cell of origin of canine peripheral T-cell lymphoma

BackgroundPeripheral T-cell lymphoma (PTCL) refers to a heterogenous group of T-cell neoplasms with poor treatment responses and survival times. Canine PTCL clinically and immunophenotypically resembles the most common human subtype, PTCL-not otherwise specified (PTCL-NOS), leading to interest in this canine disease as a naturally occurring model for human PTCL. Gene expression profiling in human PTCL-NOS has helped characterize this ambiguous diagnosis into distinct subtypes, but similar gene expression profiling in canine PTCL is lacking.MethodsBulk RNA-sequencing was performed on tumor samples from 33 dogs with either CD4+ (26/33), CD8+ (4/33), or CD4-CD8- (3/33) PTCL as diagnosed by flow cytometry, and sorted CD4+ and CD8+ lymphocytes from healthy control dogs. Following normalization of RNA-seq data, we performed differential gene expression and unsupervised clustering methods. Gene set enrichment analysis was performed to determine the enrichment of canine CD4+ PTCL for human PTCL-NOS, oncogenic pathways, and various stages of T-cell development gene signatures. We utilized gene set variation analysis to evaluate individual canine CD4+ PTCLs for various human and murine T-cell and thymocyte gene signatures. Cultured canine PTCL cells were treated with a pan-PI3K inhibitor, and cell survival and proliferation were compared to DMSO-treated controls. Expression of GATA3 and phosphorylated AKT was validated by immunohistochemistry.ResultsWhile the canine CD4+ PTCL phenotype exhibited a consistent gene expression profile, the expression profiles of CD8+ and CD4-CD8- canine PTCLs were more heterogeneous. Canine CD4+ PTCL had increased expression of GATA3, upregulation of its target genes, enrichment for PI3K/AKT/mTOR signaling, and downregulation of PTEN, features consistent with the more aggressive GATA3-PTCL subtype of human PTCL-NOS. In vitro assays validated the reliance of canine CD4+ PTCL cells on PI3K/AKT/mTOR signaling for survival and proliferation. Canine CD4+ PTCL was enriched for thymic precursor gene signatures, exhibited increased expression of markers of immaturity (CD34, KIT, DNTT, and CCR9), and downregulated genes associated with the T-cell receptor, MHC class II associated genes (DLA-DQA1, DLA-DRA, HLA-DQB1, and HLA-DQB2), and CD25.ConclusionsCanine CD4+ PTCL most closely resembled the GATA3-PTCL subtype of PTCL-NOS and may originate from an earlier stage of T-cell development than the more conventionally posited mature T-helper cell origin.

Negative Influence of Aging on Differentiation and Proliferation of CD8+ T-Cells in Dogs.

Immunosenescence is an age-related change in the immune system characterized by a reduction in naïve T-cells and an impaired proliferative capacity of CD8+ T-cells in older individuals. Recent research revealed the crucial impact of immunosenescence on the development and control of cancer, and aging is one of the causes that diminish the therapeutic efficacy of cancer immunotherapies targeting CD8+ T-cell activation. Despite dog cancer being defined as an age-related disease, there are few fundamental understandings regarding the relationship between aging and the canine immune system. Therefore, we aimed to elucidate the characteristics of immunosenescence in dogs and analyzed the effects of aging on the differentiation status and proliferation of canine CD8+ T cells using T-cell specific stimulation with anti-canine CD3/CD28 antibody-coated beads and interleukin-2. As a result, we found that older dogs have a lower proliferative capacity of CD8+ T-cells and a reduction in the naïve subset in their peripheral blood. Further analysis showed that older dogs had attenuated proliferation of the effector and central memory subsets. These results indicate the importance of maintaining less differentiated subsets to expand CD8+ T-cells in dogs and provide helpful insight into the development of dog immune therapies that require T-cell expansion ex vivo.

Development of OX40 agonists for canine cancer immunotherapy

SummaryRecent breakthroughs in cancer immunotherapy have provided unprecedented clinical benefits to human cancer patients. Cancer is also one of the most common causes of death in pet dogs. Thus, canine-specific immune therapies targeting similar signaling pathways can provide better treatment options for canine cancer patients. Here, we describe the development and characterization of two canine-specific anti-OX40 agonists to activate OX40 signaling. We show that canine OX40, like human OX40, is not expressed on resting T cells, and its expression is markedly increased on canine CD4 T cells and Tregs after stimulation with concanavalin A (Con-A). cOX40 is also expressed on tumor-infiltrating lymphocytes (TILs) in canine osteosarcoma patients. The canine-specific OX40 agonists strongly activates cPBMCs by increasing IFN-γ expression and do not require Fc receptor-mediated cross-linking for OX40 agonism. Together, these results suggest that cFcOX40L proteins are potent OX40 agonists and have the potential to enhance antitumor immunity in canine cancer patients.

Establishment and Characterization of Monoclonal Antibody Against Canine CD8 Alpha.

Understanding of the microenvironment of cancer plays a crucial role in cancer research. A tool is needed to evaluate the immune cells surrounding the cancer cells. This study establishes and evaluates a novel monoclonal antibody against canine CD8α (cCD8α). The antibody was produced by immunization of rats with cCD8α-expressing cells. After establishment and selection of hybridoma cells, the clone F3-B2 was established. The reactivity of F3-B2 was confirmed using cCD8α-overexpressing murine cells. Flow cytometric analysis also demonstrated that F3-B2 reacts with cCD8α naturally expressed in canine peripheral blood mononuclear cells and a canine T cell lymphoma cell line. The specimens of lymphoid tissue showed immunohistochemical staining for F3-B2. Moreover, we also found that F3-B2 exhibited reactivity against feline CD8. Thus, this antibody provides a good research tool to analyze CD8-positive cytotoxic lymphocytes in canine and feline tumors.

Oclacitinib induces Foxp3 expression in CD4+ T cells under activation conditions

Abstract Oclacitinib (OCL) is a novel immunosuppressive agent approved for dogs that controls inflammation in allergic disease via the inhibition of the JAK/STAT pathway. The present study attempts to shed light on the involvement of Foxp3-expressing regulatory CD4+ T (Treg) cells and inducible type 1 regulatory T (Tr1) cells in the anti-inflammatory and anti-allergic actions of OCL. Our previous study demonstrated that exposure of unstimulated canine lymphocytes to OCL did not affect Foxp3 expression in T cells. This study investigates the effect of OCL on Foxp3 expression by canine CD4+ T cells in the course of in vitro activation. Effect of OCL on generation of Tr1 (i.e. CD223+CD49b+CD4+) cells was studied using murine lymphocytes due to the lack of appropriate antibodies for the induction and detection of these cells in dogs. The exposure of canine lymphocytes to both concentration of OCL, with a concomitant stimulation, led to a significant increase in the percentage of Foxp3-expressing cells within the CD4+ T cell subset. In the light of our previous results, it can be suggested that the effect of OCL on Foxp3 expression in CD4+ T cells may depend on T cell activation status. The mean percentage of CD3/CD28-induced CD223+CD49b+CD4+ T cells was approximately 6- and 2-fold lower in the cultures exposed to OCL 10−6 and 10−7M, respectively, compared to untreated cells. Thus, the results strongly suggest that the generation of inducible Foxp3-expressing CD4+ T cells may constitute one of mechanisms responsible for the production of anti-inflammatory and anti-allergic effects of OCL. These effects are not mediated by generation of Tr1 cells. What is more, the present results indicate that OCL may counteract the induction of these cells.

Teriflunomide prevents the activation-induced CD25 expression on canine CD4+ T cells

Abstract Teriflunomide (TER) is an immunomodulatory agent. First reports on the use of TER in dogs have already appeared. Immune mechanisms underlying the immunosuppressive and anti-inflammatory properties of TER are not completely understood. The study attempts to shed light on the involvement of CD4+ T cells in inducing these properties in dogs. This study investigates the effect of TER on CD25 and Foxp3 expression by canine CD4+T cells in the course of in vitro activation. CD25 is T cell activation marker, whereas Foxp3 is critical for the development and function of CD4+ regulatory T (Treg) cells. Peripheral blood mononuclear cells collected from clinically healthy dogs (n = 15) were exposed to TER at concentrations reflecting its plasma levels obtained at a typical dose and at a ten-fold lower concentration with PMA/ionomycin stimulation. The percentage of CD25-expressing cells within CD4+ T cell subset in the culture treated with TER 10−4 M did not differ significantly from the value of this parameter in untreated non-stimulated cells but was significantly lower than that in untreated stimulated cells. The exposure to TER 10−4 M led to a significant increase in the percentage of Foxp3-expressing cells within the CD4+ T cell subset. However, the exposure to the lower concentration of TER did not affect the evaluated parameters. The results indicate that TER at the concentration achieved at a therapeutic dose prevented the activation-induced CD25 expression and induced Foxp3 expression by canine CD4+ T cells. These results suggest that the generation of inducible Foxp3-expressing CD4+ T cells and inhibition of CD4+ T cell activation may constitute one of the mechanisms responsible for the immunosuppressive and anti-inflammatory properties of TER.

Canine CD4+ T-cell lymphoma identified by flow cytometry exhibits a consistent histomorphology and gene expression profile.

T-cell lymphomas (TCL) are a diverse group of neoplasms with variable diagnostic features, pathophysiologies, therapeutic responses and clinical outcomes. In dogs, TCL includes indolent and aggressive tumours such as T-zone lymphoma (TZL) and peripheral T-cell lymphoma (PTCL), respectively. Delineation of molecular subtypes and investigation into underlying pathophysiologies of aggressive TCLs remains inadequate. We investigate the correlations between flow cytometry and histopathology of 73 cases of nodal TCL. The majority of cases (82.2%) were characterized as CD4+ TCL by flow cytometry. Fewer cases were classified as CD8+ TCL (6.8%) or CD4- CD8- TCL (11.0%). All cases, regardless of immunophenotype, exhibited conserved histologic features consistent with the WHO classification of PTCL. Histologic subsets of PTCL corresponding to immunophenotypic features were not identified. Neoplastic cell size determined by flow cytometry correlated significantly with mitotic rate. RNA-seq was performed on a subset of CD4+ PTCL cases (n = 6) and compared with sorted control CD4+ T-cells. The gene expression pattern of CD4+ PTCL was similar between all cases regardless of breed. PTCL was enriched in pathways representing G-coupled protein receptor signalling, extracellular matrix remodelling and vascular development, immune signalling and mitotic activity. Furthermore, global gene expression changes were consistent with downregulation of PTEN signalling and upregulation of the MTOR-PI3K-ATK axis. In this study, we evaluated the correlations between flow cytometry, histopathology and gene expression within a large cohort of nodal TCLs. We further demonstrate the ability of flow cytometry to identify a subtype of T-cell lymphoma, CD4+ PTCL, with a uniform histomorphology and gene expression profile.

Canine tissue-associated CD4+CD8α+ double-positive T cells are an activated T cell subpopulation with heterogeneous functional potential.

Canine CD4+CD8α+ double-positive (dp) T cells of peripheral blood are a unique effector memory T cell subpopulation characterized by an increased expression of activation markers in comparison with conventional CD4+ or CD8α+ single-positive (sp) T cells. In this study, we investigated CD4+CD8α+ dp T cells in secondary lymphatic organs (i.e. mesenteric and tracheobronchial lymph nodes, spleen, Peyer’s patches) and non-lymphatic tissues (i.e. lung and epithelium of the small intestine) within a homogeneous group of healthy Beagle dogs by multi-color flow cytometry. The aim of this systematic analysis was to identify the tissue-specific localization and characteristics of this distinct T cell subpopulation. Our results revealed a mature extrathymic CD1a-CD4+CD8α+ dp T cell population in all analyzed organs, with highest frequencies within Peyer’s patches. Constitutive expression of the activation marker CD25 is a feature of many CD4+CD8α+ dp T cells independent of their localization and points to an effector phenotype. A proportion of lymph node CD4+CD8α+ dp T cells is FoxP3+ indicating regulatory potential. Within the intestinal environment, the cytotoxic marker granzyme B is expressed by CD4+CD8α+ dp intraepithelial lymphocytes. In addition, a fraction of CD4+CD8α+ dp intraepithelial lymphocytes and of mesenteric lymph node CD4+CD8α+ dp T cells is TCRγδ+. However, the main T cell receptor of all tissue-associated CD4+CD8α+ dp T cells could be identified as TCRαβ. Interestingly, the majority of the CD4+CD8α+ dp T cell subpopulation expresses the unconventional CD8αα homodimer, in contrast to CD8α+ sp T cells, and CD4+CD8α+ dp thymocytes which are mainly CD8αβ+. The presented data provide the basis for a functional analysis of tissue-specific CD4+CD8α+ dp T cells to elucidate their role in health and disease of dogs.

Brucella canis induces canine CD4+ T cells multi-cytokine Th1/Th17 production via dendritic cell activation.

Brucella canis is a small intracellular Gram-negative bacterium that frequently leads to chronic infections highly resistant to antibiotic therapy in dogs. Also, it causes mild human brucellosis compared to other zoonotic Brucella spp. Herein we characterize the cellular immune response elicited by B. canis by analysing human and canine CD4+ T cells after stimulation with autologous monocyte-derived dendritic cells (MoDCs). Human and canine B. canis-primed MoDCs stimulated autologous CD4+ T cells; however, a Th1 response was triggered by human MoDCs, whereas canine MoDCs induced Th1/Th17 responses, with increased CD4+ T cells producing IFN-γ and IL-17A simultaneously. Each pattern of cellular response may contribute to host susceptibility, helping to understand the differences in B. canis virulence between these two hosts. In addition, other aspects of canine immunology are unveiled by highlighting the participation of IL-17A-producing canine MoDCs and CD4+ T cells producing IFN-γ and IL-17A.

A Naturally Occurring Canine Model of Peripheral T-Cell Lymphoma Not Otherwise Specified

Introduction: Peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) encompasses a heterogeneous and poorly understood group of neoplasms with poor responses to therapy and grave clinical outcomes in both canine and human patients. Because PTCL-NOS is relatively uncommon in human patients, an appropriate model is needed to facilitate elucidation of the underlying pathobiology and identification of novel therapeutic targets. A canine model of PTCL has the added potential to provide a large animal veterinary population for pre-clinical therapeutic trials and subsequent translation into human patients. Marked breed-specific predilection for this disease suggests that genetic risk factors may also be explored using the dog model.PTCL is the second most common type of lymphoma diagnosed in dogs. Similar to human patients, dogs with PTCL are usually middle-aged to older animals and typically present with peripheral lymph node involvement but systemic disease is not uncommon. Canine PTCLs are most commonly CD4-positive, often lose expression of CD5, and exhibit a histomorphology similar to that described in humans. The Boxer breed is significantly over-represented in the population of dogs that develop PTCL.Objective: In this study, we aimed to characterize the gene expression profile of canine PTCL and evaluate the molecular similarity to PTCL-NOS in human patients.Methods: RNA-sequencing was performed on lymph node aspirates from six dogs (3 Boxers and 3 non-Boxers) diagnosed with CD4+ PTCL and sorted CD4+ T-cells from control dogs. Gene expression was compared between neoplastic and non-neoplastic T cells. Gene set enrichment analysis was used to compare the gene expression profile of canine PTCL to a gene list created from a similarly designed, publically available, microarray study of human PTCL-NOS. The gene list was comprised of the top upregulated and downregulated genes in 28 cases of human PTCL-NOS when compared to sorted human control CD4+ T-cells.Results: Canine PTCL was significantly enriched for the gene set representative of human PTCL-NOS. Furthermore, similar to humans, canine PTCL upregulated pathways were involved in proliferation, cell adhesion, extracellular matrix modification, and signal transduction. Canine cases overexpressed GATA3 and CCR4; two markers which characterize a distinct subset of human PTCL-NOS associated with worse clinical outcomes. Canine cases also exhibit significant enrichment for the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) induced gene signatures, consistent with the GATA3 subgroup. Overexpression of platelet derived growth factors (PDGFRs) was conserved across both human and canine cases and has been suggested as a potential therapeutic target. GATA3 and PDGFR-alpha expression in canine cases was corroborated by immunohistochemistry. Boxers and non-Boxers exhibited markedly similar patterns of gene expression, with the exception that lymphomas from all three Boxers significantly upregulated ROS1 compared to non-Boxers.Conclusions: From this work we conclude that canine CD4+ PTCL is molecularly similar to an aggressive subset of human PTCL-NOS and may serve as a useful model to further investigate the molecular drivers of this aggressive and poorly understood disease. DisclosuresNo relevant conflicts of interest to declare.

Oclacitinib depletes canine CD4+ and CD8+ T cells in vitro

Oclacitinib (OCL) is a novel immunosuppressive agent approved for dogs that controls itch and inflammation in allergic disease via the inhibition of the JAK/STAT pathway. This paper investigates the in vitro effect of OCL, a novel Janus kinase inhibitor, on selected canine regulatory (Treg) and effector (Teff) CD4+ and CD8+ T cells. Exposure of peripheral blood lymphocytes to OCL did not affect the transcription factor Foxp3 (Forkhead Box P3 protein) expression in CD25+CD4+ and CD25+CD8+ T cells. Moreover, OCL did not influence constitutive CD25 expression on these cells although it reduced the activation-induced CD25 expression on CD4+ T cells. Unexpectedly, the research demonstrated the cytoreductive and proapoptotic effects of OCL on the cells examined. Exposure to OCL caused a dramatic loss of both CD4+ and CD8+ T cells and this effect was observed in both Treg and Teff cell subsets. On the one hand, cytoreductive and proapoptotic effects of OCL toward CD4+ and CD8+ Teff cells, as well as the drug-induced inhibition of CD4+ T cell activation, may be considered as additional mechanisms involved in producing anti-inflammatory and anti-allergic properties of the drug. On the other hand, these effects also represent the immunosuppressive action in the sense of an unwanted effect because CD4+ and CD8+ Teff cells play a crucial role in the production of cellular immunity. Further studies are needed to determine whether the use of OCL actually creates the risk of such action.

Canine CD4+CD8+ double-positive T cells participate in immunity to Ehrlichia chaffeensis infection

Abstract Ehrlichia chaffeensis is an obligate intracellular gram-negative bacteria and a frequent cause of severe and fatal tick-borne infection in people in North America. The reservoir host for E. chaffeensis is the white-tailed deer, while humans, dogs, coyotes and goats are regarded as common incidental hosts. In dogs, we have shown that E. chaffeensis establishes a chronic infection that persists for at least 42 days, while promoting development of Th1 and Th17 cellular responses and pathogen-specific humoral immunity. To date, little is known about the immune responses that correlate with control or successful clearance of the infection in dogs or humans. In dogs, a population of mature CD4+CD8+ double-positive (DP) T cells exist that have features of activated T cells and share some similarities to the CD4+CD8+ DP populations which have been described in pigs and humans. However, little is known about the function of these cells in dogs, particularly in the context of infectious diseases. Here, we demonstrate that canine CD4+CD8+ DP T cells expand significantly in the blood and spleen in response E. chaffeensis infection, and upregulate markers of T cell activation and antigen-experience. Recall responses after in vitro restimulation with E. chaffeensis host-cell free lysate showed that CD4+CD8+ DP T cells undergo multiple rounds of antigen-specific proliferation, produce IFNγ and IL-17, and upregulate expression of the cytotoxic molecules granzyme B and granulysin. Together, our results demonstrate that CD4+CD8+ DP T cells contribute to the immune response to E chaffeensis in the physiologic host, and suggest the importance of alternative lymphocyte populations in promoting protection and control of tick-borne diseases in the dog.

Canine peripheral blood CD4+CD8+ double-positive T cell subpopulations exhibit distinct T cell phenotypes and effector functions

Canine peripheral blood CD4+CD8α+ double-positive (dp) Tcells represent a small population of T lymphocytes that are not only derived from single-positive (sp) CD4+ but also from CD8+ Tcells. Dp Tcells can be subdivided in three different subpopulations according to their expression levels of CD4 and CD8α, i.e. the CD4dimCD8αbright, CD4brightCD8αbright, and the CD4brightCD8αdim subsets. Previously we could show that total canine peripheral CD4+CD8α+ dp Tcells have features of activated Tcells. Here we demonstrate that the individual dp Tcell subsets distinctly differ in their activation status and phenotype. Thus, among the dp Tcell subsets the CD4dimCD8αbright subpopulation has the lowest and the CD4brightCD8αbright subset the highest frequency of CD25+ cells pointing to the CD4brightCD8αbright subset with the highest activation status. Consistent with that, analysis of CD44 vs. CD62L expression demonstrates that the CD4brightCD8αbright subset contains the highest frequencies of effector-memory Tcells (TEM). Upon in vitro stimulation, the CD4brightCD8αbright and CD4dimCD8αbright dp Tcell subsets show the highest frequencies of IFN-γ producing cells. Expression of granzyme B was found exclusively in the CD4dimCD8αbright dp Tcell subset indicating cytotoxic potential of this dp Tcell subset. Furthermore, T-bet expression was restricted to the CD4dimCD8αbright subset, while Foxp3+ regulatory Tcells were detected in the CD4brightCD8αdim dp Tcell subset. In conclusion, canine dp Tcells are phenotypically and functionally heterogeneous consistent with the diverse characteristics of their single-positive CD4+ and CD8+ Tcell progenitors. The data provide the basis for future in vivo studies to elucidate the role of individual dp Tcell subsets in host defense and/or immune pathological diseases of dogs.

Peripheral canine CD4+CD8+ double-positive T cells – unique amongst others

T lymphocytes co-expressing CD4 and CD8 (“double-positive T cells”) are commonly associated with a thymic developmental stage of T cells. Their first description in humans and pigs as extrathymic T cells with a memory phenotype almost 30 years ago came as a surprise. Meanwhile peripheral double-positive T cells have been described in a growing number of different species. In this review we highlight novel data from our very recent studies on canine peripheral double-positive T cells which point to unique features of double-positive T cells in the dog. In contrast to porcine CD4+CD8+ T cells forming a homogenous cellular population based on their expression of CD4 and CD8α, canine CD4+CD8+ T cells can be divided into three different cellular subsets with distinct expression levels of CD4 and CD8α. Double-positive T cells expressing CD8β are present in humans and dogs but absent in swine. Moreover, canine CD4+CD8+ T cells can not only develop from CD4+ single-positive T cells but also from CD8+ single-positive T cells. Together, this places canine CD4+CD8+ T cells closer to their human than porcine counterparts since human double-positive T cells also appear to be heterogeneous in their CD4 and CD8α expression and have both CD4+ and CD8+ T cells as progenitor cells. However, CD4+ single-positive T cells are the more potent progenitors for canine double-positive T cells, whereas CD8+ single-positive T cells are more potent progenitors for human double-positive T cells. Canine double-positive T cells have an activated phenotype and may have as yet unrecognized roles in vivo in immunity to infection or in inflammatory diseases such as chronic infection, autoimmunity, allergy, or cancer.

Stimulation with Concanavalin-A Induces IL-17 Production by Canine Peripheral T Cells.

The characteristics of canine IL-17-producing cells are incompletely understood. Expression of mRNA encoding orthologs of IL-17 and the IL-17 receptor has been documented in tissues from dogs with arthritis, inflammatory bowel disease, and lymphoma; however, no associations have been found between IL-17 gene expression and disease phenotype in these conditions. Robust assessment of the role of IL-17-producing cells in dogs will require measuring the frequency of these cells in health and disease in balance with other lymphocyte subsets. The aim of this study was to confirm that the T-cell IL-17 response in dogs is evolutionarily conserved. Canine peripheral blood mononuclear cells were stimulated with Concanavalin A with or without polarizing cytokines. We used a canine specific IL-17 ELISA and flow cytometry to identify IL-17-producing T cells. Accumulation of intracellular IL-17 was observed in stimulated CD4 and CD8 T cells. The addition of pro-inflammatory cytokines appeared to enhance polarization of canine CD4 T cells to the Th17 phenotype. Conversely, the addition of IL-2 in the presence of TGF-β resulted in expansion of Treg cells. We conclude that canine IL-17-producing cells behave similarly to those from humans and mice when stimulated with mitogens and polarized with pro-inflammatory or immune regulatory cytokines.