The term histiocyte has been used to generically describe cells of dendritic cell (DC) or macrophage lineage. Dendritic cells are the most potent antigen-presenting cells (APCs) for induction of immune responses in naive T cells. Aspects of the developmental and migratory program of DCs are recapitulated in the section on Canine histiocytic diseases. To classify hematopoietic and lymphoid neoplasia according to the World Health Organization (WHO) system as applied to the domestic species, it is important to have access to markers for immunohistochemical (IHC) analysis. Determination of cell lineage is a critical problem in classification of leukocytic neoplasia. Histiocytoma is a common, benign, cutaneous neoplasm of the dog. Cutaneous Langerhans cell histiocytosis (LCH) is well documented in humans. Pulmonary LCH is a disease of aged cats, which causes progressive respiratory failure leading to euthanasia. Feline progressive histiocytosis (FPH) is a disease of middle-aged to older cats, the age ranging from 7 to 17 years.

BACKGROUND:Erythema multiforme (EM) and similar cytotoxic dermatoses, including Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), represent immune-mediated conditions that have clinical, histopathological and immunohistochemical overlap with other diseases. Although reactive processes are typically associated with polyclonal expansion of lymphocyte populations, benign clonal expansion is possible in non-neoplastic conditions. HYPOTHESIS/OBJECTIVES:The purpose of this study is to elucidate the role of clonality in differentiating cases of canine EM/SJS/TEN from cutaneous epitheliotropic lymphoma. Further aims include providing clinical correlation and response to therapy in combination with clonality. It is hypothesised that both clonal and polyclonal expansions will be observed in cases of EM/SJS/TEN. ANIMALS:Twelve dogs with clinical and histopathological changes supportive of EM or SJS/TEN. MATERIALS AND METHODS:Clinical data, histological and immunohistochemical examination as well as clonality for T-cell receptor gamma (TRG) was performed for tissue samples in canine EM/SJS/TEN. Modified drug scoring was performed for cases with medication administration before lesion development. RESULTS:Twelve cases were included for retrospective review. Good response to therapy, CD3 immunoreactive T cells, and at least minor expression of Granzyme B were noted in all cases. Eleven of 12 had mild-to-moderate CD20 dermal infiltration. Polyclonal populations were noted in four cases, polyclonal with minor clones in five cases and clonality in three cases. Modified drug scoring was positive in five of six cases. CONCLUSIONS AND CLINICAL RELEVANCE:This study describes cases of canine EM/SJS/TEN demonstrating both polyclonal and clonal T-cell expansion, further highlighting the need for pairing clinical response with histopathological results and advanced diagnostics.

An 8-y-old, spayed female Bernese Mountain Dog was presented to a referral center for evaluation of right thoracic limb lameness and previously suspected Evans syndrome that had been poorly responsive to immunosuppressive therapy. Based on review of examination findings and laboratory data, Evans syndrome was deemed unlikely and hemophagocytic histiocytic sarcoma (HHS) was strongly suspected. On blood smear evaluation, atypical, histiocytic cells were noted, some of which exhibited siderophagia. Considering that circulating cells are not typically observed in dogs with HHS, additional diagnostic investigation was performed. Autopsy and histopathology revealed that the dog had a mixed form of HS (dendritic-cell origin HS in the lung, and HHS in the spleen, liver, and bone marrow), and immunocytochemical characterization of cultured cells derived from blood suggested that the cells were of dendritic HS origin, rather than HHS origin, as originally suspected. Whole-exome sequencing revealed genetic similarity between cell lines derived from lung tissue and blood, providing additional evidence of the relatedness of these 2 cell populations. Our case highlights the rare entity of mixed HS and typifies the inherent challenges in classifying rare, atypical, circulating neoplastic cells.

The dog is an important companion animal and also serves as model species for human diseases. Given the central role of T cells in immune responses, a basic understanding of canine conventional T cell receptor (TCR)αβ+ T cells, comprising CD4+ single-positive (sp) T helper (Th) and CD8α+ sp cytotoxic T cell subsets, is available. However, characterization of canine non-conventional TCRαβ+ CD4+CD8α+ double-positive (dp) and TCRαβ+ CD4−CD8α− double-negative (dn) T cells is limited. In this study, we performed a comprehensive analysis of canine dp and dn T cells in comparison with their conventional counterparts. TCRαβ+ T cells from peripheral blood of healthy dogs were sorted according to their CD4/CD8α phenotype into four populations (i.e. CD4+ sp, CD8α+ sp, dp, and dn) and selected surface markers, transcription factors and effector molecules were analyzed ex vivo and after in vitro stimulation by RT-qPCR. Novel characteristics of canine dp T cells were identified, expanding the previously characterized Th1-like phenotype to Th17-like and Th2-like properties. Overall, mRNA expression of various Th cell-associated cytokines (i.e. IFNG, IL17A, IL4, IL13) in dp T cells upon stimulation highlights their versatile immunological potential. Furthermore, we demonstrated that the CD4-CD8α- dn phenotype is stable during in vitro stimulation. Strikingly, dn T cells were found to express highest mRNA levels of type 2 effector cytokines (IL4, IL5, and IL13) upon stimulation. Their strong ability to produce IL-4 was confirmed at the protein level. Upon stimulation, the percentage of IL-4-producing cells was even higher in the non-conventional dn than in the conventional CD4+ sp population. Constitutive transcription of IL1RL1 (encoding IL-33Rα) further supports Th2-like properties within the dn T cell population. These data point to a role of dn T cells in type 2 immunity. In addition, the high potential of dn T cells to transcribe the gene encoding the co-inhibitory receptor CTLA-4 and to produce the inhibitory cytokine IL-10 indicates putative immunosuppressive capacity of this population. In summary, this study reveals important novel aspects of canine non-conventional T cells providing the basis for further studies on their effector and/or regulatory functions to elucidate their role in health and disease.

This report describes a multicentric intermediate-size B-cell lymphoma with epitheliotropism in a Freiberger mare affecting multiple mucous membranes, skin and internal organs. The clonal neoplastic B-cell population was accompanied by numerous reactive polyclonal small T cells. Differential diagnoses for these unusual findings are discussed. Xxxxxxxx. Xxxxxxxx. Xxxxxxxx. This report describes a multicentric intermediate-size B-cell lymphoma with epitheliotropism in a Freiberger mare affecting multiple mucous membranes, skin and internal organs. The clonal neoplastic B-cell population was accompanied by numerous reactive polyclonal small T cells. Differential diagnoses for these unusual findings are discussed.image

Feline chronic enteropathy is a poorly defined condition of older cats that encompasses chronic enteritis to low-grade intestinal lymphoma. The histological evaluation of lymphocyte numbers and distribution in small intestinal biopsies is crucial for classification and grading. However, conventional histological methods for lymphocyte quantification have low interobserver agreement, resulting in low diagnostic reliability. This study aimed to develop and validate an artificial intelligence (AI) model to detect intraepithelial and lamina propria lymphocytes in hematoxylin and eosin-stained small intestinal biopsies from cats. The median sensitivity, positive predictive value, and F1 score of the AI model compared with the majority opinion of 11 veterinary anatomic pathologists, were 100% (interquartile range [IQR] 67%-100%), 57% (IQR 38%-83%), and 67% (IQR 43%-80%) for intraepithelial lymphocytes, and 89% (IQR 71%-100%), 67% (IQR 50%-82%), and 70% (IQR 43%-80%) for lamina propria lymphocytes, respectively. Errors included false negatives in whole-slide images with faded stain and false positives in misidentifying enterocyte nuclei. Semiquantitative grading at the whole-slide level showed low interobserver agreement among pathologists, underscoring the need for a reproducible quantitative approach. While semiquantitative grade and AI-derived lymphocyte counts correlated positively, the AI-derived lymphocyte counts overlapped between different grades. Our AI model, when supervised by a pathologist, offers a reproducible, objective, and quantitative assessment of feline intestinal lymphocytes at the whole-slide level, and has the potential to enhance diagnostic accuracy and consistency for feline chronic enteropathy.

IntroductionThe domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing.MethodsPeripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, αβ T cell receptor transcripts and γδ T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5’ reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs.ResultsUnsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and γδ T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis identified a subset of CD8+ cytotoxic T cells with skewed TRA and TRB gene usage, conserved TRA and TRB junctional motifs, restricted TRA/TRB pairing and reduced diversity in TRG junctional length. We also identified a public γδ T cell subset with invariant TRD and TRG chains and a CD4+ TEM-like phenotype. Among monocytic cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster.DiscussionOur study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

A dog presented with deep pyoderma on the paw, following treatment with ciclosporin and prednisone for immune-mediated haemolytic anaemia. Cytological evaluation, skin biopsy, aerobic culture, next-generation DNA sequencing and PCR were used to detect the first reported case of Burkholderia gladioli in a dog.

Conventional CD4pos regulatory T (Treg) cells characterized by expression of the key transcription factor forkhead box P3 (FoxP3) are crucial to control immune responses, thereby maintaining homeostasis and self-tolerance. Within the substantial population of non-conventional T cell receptor (TCR)αβpos CD4negCD8αneg double-negative (dn) T cells of dogs, a novel FoxP3pos Treg-like subset was described that, similar to conventional CD4pos Treg cells, is characterized by high expression of CD25. Noteworthy, human and murine TCRαβpos regulatory dn T cells lack FoxP3. Immunosuppressive capacity of canine dn T cells was hypothesized based on expression of inhibitory molecules (interleukin (IL)-10, cytotoxic T-lymphocyte associated protein 4, CTLA4). Here, to verify their regulatory function, the dnCD25pos (enriched for FoxP3pos Treg-like cells) and the dnCD25neg fraction, were isolated by fluorescence-activated cell sorting from peripheral blood mononuclear cells (PBMC) of Beagle dogs and analyzed in an in vitro suppression assay in comparison to conventional CD4posCD25pos Treg cells (positive control) and CD4posCD25neg T cells (negative control). Canine dnCD25pos T cells suppressed the Concanavalin A-driven proliferation of responder PBMC to a similar extent as conventional CD4posCD25pos Treg cells. Albeit to a lesser extent than FoxP3-enriched dn and CD4posCD25pos populations, even dnCD25neg T cells reduced the proliferation of responder cells. This is remarkable, as dnCD25neg T cells have a FoxP3neg phenotype comparable to non-suppressive CD4posCD25neg T cells. Both, CD25pos and CD25neg dn T cells, can mediate suppression independent of cell-cell contact and do not require additional signals from CD4posCD25neg T cells to secrete inhibitory factors in contrast to CD4posCD25pos T cells. Neutralization of IL-10 completely abrogated the suppression by dnCD25pos and CD4posCD25pos Treg cells in a Transwell™ system, while it only partially reduced suppression by dnCD25neg T cells. Taken together, unique canine non-conventional dnCD25pos FoxP3pos Treg-like cells are potent suppressor cells in vitro. Moreover, inhibition of proliferation of responder T cells by the dnCD25neg fraction indicates suppressive function of a subset of dn T cells even in the absence of FoxP3. The identification of unique immunoregulatory non-conventional dn T cell subpopulations of the dog in vitro is of high relevance, given the immunotherapeutic potential of manipulating regulatory T cell responses in vivo.

Supplementary Figure from B7-H3 Specific CAR T Cells for the Naturally Occurring, Spontaneous Canine Sarcoma Model

Feline pulmonary Langerhans cell histiocytosis (FPLCH) is a rare histiocytic proliferative disease of middle-aged to older domestic cats. Langerhans cells in the terminal airways proliferate and infiltrate the interstitium and the airways to a lesser degree, widely effacing normal parenchyma. Historically, definitive diagnosis has required postmortem evaluation where pulmonary lesions have a classic gross and histologic morphology. Here, we present the first documented antemortem diagnosis of FPLCH using bronchoalveolar lavage (BAL) cytology and immunocytochemistry (ICC) in a 9-year-old British shorthair mix. The cat had a 3-month history of respiratory difficulty that was refractory to steroids and antimicrobials. Pulmonary radiographs had marked diffuse changes with a complex bronchointerstitial and micronodular pattern. BAL cytology revealed neutrophilic inflammation and markedly increased histiocytes with morphology distinct from typical pulmonary macrophages. ICC characterized histiocytes as CD1a+ /E-cadherin+ /CD11b- /PanCK- , consistent with a Langerhans cell phenotype. The cat was humanely euthanized due to poor prognosis and presented for necropsy. Gross, histopathologic, immunophenotypic, and ultrastructural findings confirmed a diagnosis of FPLCH. Proliferative cells were E-cadherin+ /Iba-1+ /CD18+ /CD1a+ /CD5+ /MHCII+ /CD204- /CD4- ; transmission electron microscopy identified the presence of Birbeck granules in the proliferating histiocytes, consistent with previous reports of FPLCH.

BACKGROUND:The antitumour effects of interferon-gamma (IFN-γ) in humans with cutaneous epitheliotropic T-cell lymphoma (CETCL) have been described; however, the efficacy of IFN-γ in dogs has not been investigated.HYPOTHESIS/OBJECTIVES:The aim of this study was to evaluate the efficacy of recombinant canine IFN-γ (rCaIFN-γ) therapy in dogs with CETCL.ANIMALS:Twenty dogs with CETCL recruited from seven veterinary clinics were enrolled in the study.MATERIALS AND METHODS:Fifteen dogs were treated with rCaIFN-γ, and five control dogs were treated with prednisolone. We evaluated survival time, skin lesions (erythema, nodules, ulcers and bleeding), pruritus and general condition (sleep, appetite and body weight). In the rCaIFN-γ group, a questionnaire regarding the therapy was administered to owners after the dogs died.RESULTS:No significant differences existed in the median survival time between the rCaIFN-γ and control groups (log-rank test: p = 0.2761, Wilcoxon's rank sum test: p = 0.4444). However, there were significant differences in ulcer, bleeding, pruritus, sleep, appetite and body weight between the groups (Wilcoxon-Mann-Whitney U-test: p = 0.0023, p = 0.0058, p = 0.0005, p = 0.0191, p = 0.0306 and p = 0.0306, respectively). Two (40%) of five dogs were euthanised in the control group, compared with none in the rCaIFN-γ group. Fourteen questionnaires were collected, and owners reported that they were satisfied with the rCaIFN-γ treatment.CONCLUSIONS AND CLINICAL RELEVANCE:Although the median survival time was not prolonged, rCaIFN-γ could be helpful in maintaining good quality of life for dogs with CETCL.

Canine cutaneous histiocytomas originate from Langerhans cells. Multiple histiocytomas are referred to as cutaneous Langerhans cell histiocytosis. Feline pulmonary Langerhans cell histiocytosis causes respiratory failure owing to extensive lung infiltration. Localized and disseminated histiocytic sarcomas usually arise from interstitial dendritic cells. Primary sites include spleen, lung, skin, brain (meninges), lymph node, bone marrow, and synovial tissues of limbs. An initially indolent form of localized histiocytic sarcomas, progressive histiocytosis, originates in the skin of cats. Hemophagocytic histiocytic sarcomas originates in splenic red pulp macrophages. Canine reactive histiocytoses (systemic histiocytosis and cutaneous histiocytosis) are complex inflammatory diseases with underlying immune dysregulation.

The dog is valued as a companion animal and increasingly recognized as a model for human disorders. Given the importance of T cells in health and disease, comprehensive knowledge of canine T cells can contribute to our understanding of pathogenesis mechanisms and inform the development of new treatment strategies. However, the diversity of canine T cells is still poorly understood mainly due to the lack of species-reactive antibodies for use in flow cytometry. The aim of this study was to generate a detailed atlas of peripheral blood TCRαβ + T cells of healthy dogs using single-cell RNA-sequencing (scRNAseq) combined with immune repertoire sequencing. A total of 22 TCRαβ + T cell clusters were identified, which were classified into three major groups: CD4-dominant (11 clusters), CD8A-dominant (8 clusters), and CD4/CD8A-mixed (3 clusters). Based on differential gene expression, distinct differentiation states (naïve, effector, memory, exhausted) and lineages (e.g. CD4 T helper and regulatory T cells) could be distinguished. Importantly, several T cell populations were identified, which have not been described in dogs before. Of particular note, our data provide first evidence for the existence of canine mucosa-associated invariant T cell (MAIT)-like cells, representing one of three newly identified FCER1G + innate-like CD8A + T cell populations in the peripheral blood of healthy dogs. In conclusion, using scRNAseq combined with immune repertoire sequencing we were able to resolve canine TCRαβ + T cell populations at unprecedented resolution. The peripheral blood TCRαβ + T cell atlas of healthy dogs generated here represents an important reference data set for future studies and is of relevance for identifying new targets for T cell-specific therapies.

Epizootic bovine abortion (EBA) is an arthropod-borne bacterial disease that causes significant economic loss for cattle producers in the western United States. The etiologic agent, Pajaroellobacter abortibovis, is an intracellular pathogen that has yet to be cultivated in vitro, thereby requiring novel methodologies for vaccine development. A vaccine candidate, using live P. abortibovis-infected cells (P.a-LIC) harvested from mouse spleens, was tested in beef cattle. Over the course of two safety studies and four efficacy trials, safety risks were evaluated, and dosage and potencies refined. No incidence of anaphylaxis, recognized health issues or significant impact upon conception rates were noted. Vaccination did result in subclinical skin reactions. Early fetal losses were noted in two trials and were significant when the vaccine was administered within 21 days prior to conception. Administration of the EBA agent (EBAA) vaccine as a single dose, at a potency of 500 P.a–LIC, 56 days prior to breeding, provided 100% protection with no early fetal losses. Seroconversion occurred in all animals following EBAA vaccination and corresponded well with protection of the fetus from epizootic bovine abortion.

Background Cutaneous epitheliotropic T-cell lymphoma is a malignant tumour of the skin already reported in humans, dogs, cats, horses, and other species, but not previously in donkeys. The standard diagnosis is based on clinical, morphological and immunophenotypic data. Differentiation of malignant versus benign proliferation of lymphocytes is crucial; in ambiguous cases T-cell receptor gamma (TRG) molecular clonality should be tested. In the present paper, we report a case of mycosis fungoides diagnosed in a donkey whose diagnosis was based on clinical, histological and immunohistochemical aspects and a positive TRG clonality test. Case presentation A twenty-five-year-old donkey gelding was referred with a mildly pruritic, generalised and severe exfoliative dermatosis. Otherwise, the animal was clinically healthy, though mildly underweight. Dermatological examination revealed severe generalised alopecic and exfoliative dermatitis, occasionally eroded, with high number of large, thin, greyish scales. All mucocutaneous junctions except the hoofs were affected. Ectoparasites and dermatophytes were ruled out. The complete blood count and blood smear evaluation revealed mild normocytic normochromic anemia. The biochemistry panel showed mild hyperproteinemia with albumin within the normal range. Protein electrophoresis showed moderate polyclonal hypergammaglobulinemia. Histological findings were characterised by interface dermatitis with massive exocytosis in the epidermis of a homogenous population of lymphoid cells showing atypia. Clusters of neoplastic cells were present within the epidermis forming Pautrier “microabscesses”. These findings are consistent with cutaneous epitheliotropic lymphoma. Immunohistochemical staining revealed uniform labelling of the neoplastic cells for CD3, and lack of expression of CD20 (a B cell lineage associated marker). Molecular clonality PCR (PARR) was performed using equine TRG primers; this revealed a clonal rearrangement in a heavy polyclonal background. Transmission electronic microscopy showed multiple lymphocytes with convoluted or cerebriform nuclei. Conclusions This case report provides the first evidence of clinical, histopathological, immunophenotypic features, electron microscopy findings and molecular analysis of a cutaneous epitheliotropic T-cell lymphoma (mycosis fungoides) in a donkey. Our observations suggest that cutaneous T-cell lymphoma should be included in the differential diagnoses of exfoliative dermatitis, even those progressing in a chronic pattern and/or with few or no pruritus.

Abstract One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant animal models. In this study, we sought to establish a chimeric antigen receptor (CAR) T-cell treatment model for naturally occurring canine sarcomas as a model for human CAR T-cell therapy. Canine CARs specific for B7-H3 were constructed using a single-chain variable fragment derived from the human B7-H3–specific antibody MGA271, which we confirmed to be cross-reactive with canine B7-H3. After refining activation, transduction, and expansion methods, we confirmed target killing in a tumor spheroid three-dimensional assay. We designed a B7-H3 canine CAR T-cell and achieved consistently high levels of transduction efficacy, expansion, and in vitro tumor killing. Safety of the CAR T cells were confirmed in two purposely bred healthy canine subjects following lymphodepletion by cyclophosphamide and fludarabine. Immune response, clinical parameters, and manifestation were closely monitored after treatments and were shown to resemble that of humans. No severe adverse events were observed. In summary, we demonstrated that similar to human cancers, B7-H3 can serve as a target for canine solid tumors. We successfully generated highly functional canine B7-H3–specific CAR T-cell products using a production protocol that closely models human CAR T-cell production procedure. The treatment regimen that we designed was confirmed to be safe in vivo. Our research provides a promising direction to establish in vitro and in vivo models for immunotherapy for canine and human solid tumor treatment.

BACKGROUND:Canine epitheliotropic cutaneous T-cell lymphoma (eCTCL) is thought to represent a disease homologue to human mycosis fungoides (MF). In human MF, neoplastic cells are phenotypically consistent with resident effector memory T cells, a population that remains for an extended period within tissue without circulating. Dogs with eCTCL often present with lesions in multiple locations, raising the question of whether the neoplasm is of the same T-cell subpopulation or not.OBJECTIVES:To characterize the antigen receptor gene rearrangements of lymphocytes from skin and blood of dogs with eCTCL to determine if neoplastic clones are identical.ANIMALS:Fourteen dogs with eCTCL.MATERIALS AND METHODS:Histological and immunohistochemical examination, and PCR for antigen receptor rearrangement (PARR) for T-cell receptor gamma (TRG) performed on multiple cutaneous biopsy samples and blood.RESULTS:All skin biopsies contained cluster of differentiation (CD)3-positive neoplastic lymphocytes. Within individual dogs, all skin biopsies revealed identical TRG clonality profiles, suggesting that the same neoplastic clone was present in all sites. In the blood, a matching clone was found in six of 14 dogs, a unique clone was observed in nine of 14 dogs, and no clone was detected in two of 14 dogs.CONCLUSIONS:These findings show that canine eCTCL lesions in multiple locations harbour the same neoplastic clone, neoplastic lymphocytes do not remain fixed to the skin and instead can circulate via blood, differing clones can be identified in skin versus blood, and circulating neoplastic cells can be detected without lymphocytosis.

Human natural killer (NK) cells can target tumor cells in an antigen-specific manner by the recognition of cell bound antibodies. This process induces antibody-dependent cell-mediated cytotoxicity (ADCC) and is exclusively mediated by the low affinity IgG Fc receptor CD16A (FcγRIIIA). Exploiting ADCC by NK cells is a major area of emphasis for advancing cancer immunotherapies. CD64 (FcγRI) is the only high affinity IgG FcR and it binds to the same IgG isotypes as CD16A, but it is not expressed by human NK cells. We have generated engineered human NK cells expressing recombinant CD64 with the goal of increasing their ADCC potency. Preclinical testing of this approach is essential for establishing efficacy and safety of the engineered NK cells. The dog provides particular advantages as a model, which includes spontaneous development of cancer in the setting of an intact and outbred immune system. To advance this immunotherapy model, we cloned canine CD16A and CD64 and generated specific mAbs. We report here for the first time the expression patterns of these FcγRs on dog peripheral blood leukocytes. CD64 was expressed by neutrophils and monocytes, but not lymphocytes, while canine CD16A was expressed at high levels by a subset of monocytes and lymphocytes. These expression patterns are similar to that of human leukocytes. Based on phenotypic characteristics, the CD16A+ lymphocytes consisted of T cells (CD3+ CD8+ CD5dim α/β TCR+) and NK cells (CD3− CD5− CD94+), but not B cells. Interestingly, the majority of canine CD16A+ lymphocytes were from the T cell population. Like human CD16A, canine CD16A was downregulated by a disintegrin and metalloproteinase 17 (ADAM17) upon leukocyte activation, revealing a conserved means of regulation. We also directly demonstrate that both canine CD16A and CD64 can induce ADCC when expressed in the NK cell line NK-92. These findings pave the way to engineering canine NK cells or T cells with high affinity recombinant canine CD64 to maximize ADCC and to test their safety and efficacy to benefit both humans and dogs.