Commercially available rabbit anti-human polyclonal antisera as a useful tool for immune system studies in veterinary species

We have used selected rabbit anti-human polyclonal antibodies as an example of useful and easily available tools for studies on immune system structure and development in important veterinary species, many of which also represent animal models in biomedicine. The cocktail of anti-human Igkappa-FITC/anti-Iglambda-RPE F(ab')(2) fragments was used for two-colour and, in combination with the cross-reactive anti-CD79alpha monoclonal antibody HM-57, for three-colour flow cytometry of canine, feline, bovine and porcine peripheral B-cells. A possible application of such immunoreagents in studies on primary B-cell differentiation has been suggested in pigs; the same approach can be used in other species of interest. Rabbit anti-human lactoferrin-FITC F(ab')(2) fragment was used for visualizing neutrophils in dogs, pigs and cattle and an application for two-colour immunophenotyping of canine granulocyte subsets has been designed. Affinity isolated rabbit anti-human CD3 and anti-human TdT have been shown to represent a ready-to-use tool for in situ studies on primary T-lymphopoiesis in pigs with possible extensions both to the B-lineage development in pigs and other animal models. Altogether, our study show that carefully selected polyclonal antibodies available on the market may possess broad cross-reactivity with important applications in veterinary research.     

Suppression of canine myeloid cells by soluble factors from cultured canine tumor cells

BackgroundCancer profoundly affects immunity and causes immunosuppression that contributes to tumor escape, metastases and resistance to therapy. The mechanisms by which cancer cells influence immune cells are not fully known but both innate and adaptive immune cells can be altered by cancer. Myeloid cells are innate immune cells that comprise the mononuclear phagocytic system (MPS) and include monocytes, macrophages, dendritic cells (DCs) and their progenitors. Myeloid cells play important roles in both the promotion and regulation of immune responses. Dysregulated myeloid cells are increasingly being recognized as contributing to cancer-related immunosuppression. This study investigated whether soluble factors produced by canine tumor cells inhibited canine myeloid cell function.MethodsThese studies investigated the utility of using the canine DH82 cell line for assessment of canine myeloid responses to tumor-derived soluble factors (TDSFs). Phenotypic comparisons to canine bone marrow-derived DCs (BM-DCs) and bone marrow-derived macrophages (BM-MΦs) were performed and expression of myeloid cell markers CD11b, CD11c, CD80, and major histocompatibility complex (MHC) class II were evaluated by flow cytometry. Phenotypic and functional changes of DC populations were then determined following exposure to tumor-conditioned media (TCM) from canine osteosarcoma, melanoma and mammary carcinoma cell lines.ResultsWe found that the canine BM-DCs and the DH82 cell line shared similar CD11b, CD11c and MHC II expression and morphologic characteristics that were distinct from canine BM-MΦs. Myeloid cells exposed to TDSFs showed decreased expression of MHC class II and CD80, had reduced phagocytic activity and suppressed the proliferation of responder immune cells.ConclusionThese results show that soluble factors secreted from canine tumor cells suppress the activation and function of canine myeloid cells. Our results suggest that, similar to humans, dysregulated myeloid cells may contribute to immunosuppression in dogs with cancer.     

Species-specific properties and translational aspects of canine dendritic cells

Dogs are affected by spontaneously occurring neoplastic and inflammatory diseases which often share many similarities with pathological conditions in humans and are thus appreciated as important translational animal models. Dendritic cells (DCs) represent the most potent antigen presenting cell population. Besides their physiological function in the initiation of primary T cell responses and B cell immunity, a deregulation of DC function is involved in immune-mediated tissue damage, immunosuppression and transplantation complication in human and veterinary medicine. DCs represent a promising new target for cancer immunotherapy in dogs. However, the therapeutic use of canine DCs is restricted because of a lack of standardized isolation techniques and limited information about dog-specific properties of this cell type. This article reviews current protocols for the isolation and in vitro generation of canine monocyte- and bone marrow-derived DCs. DCs of dogs are characterized by unique morphological features, such as the presence of cytoplasmic projections and periodic microstructures. Canine DCs can be discriminated from other hematopoietic cells also based on phenotypic properties and their high T cell stimulatory capability in mixed leukocyte reactions. Furthermore, the classification of canine DC-derived neoplasms and the role of DCs in the pathogeneses of selected infectious, allergic and autoimmune diseases, which share similarities with human disorders, are discussed. Future research is needed to expand the existing knowledge about DC function in canine diseases as a prerequisite for the development of future therapies interfering with the immune response.     

Pulmonary dendritic cells isolated from neonatal and adult ovine lung tissue

Lung dendritic cells (DCs) are potent antigen presenting cells (APCs) that initiate and modulate the adaptive immune response upon microbial infection within the pulmonary environment. For the first time, neonatal and adult lung DCs in a large animal model were compared in these studies. Here, we isolated and identified lung DCs in both neonatal and adult sheep, a valuable experimental animal utilized in pulmonary studies of naturally occurring respiratory diseases. Neonatal lung DCs exhibited characteristic dendrites and morphology when observed by transmission electron microscopy and expressed low to moderate DEC-205, CD80/86, MHC class II and CD 14. Regardless of age, lung DCs were functionally able to endocytose FITC conjugated ovalbumin but to a lesser degree than monocyte-derived DCs. In addition, neonatal lung DCs were demonstrated to be potent stimulators of allogeneic T cell proliferation. Together, these results demonstrate that neonatal and adult lung DCs are functionally similar. It is apparent from the data presented that neonatal pulmonary DCs do not exhibit an intrinsic functional defect that would impair their ability to take up antigen and stimulate na?ve T cells. These data support growing evidence that neonatal immune responses may differ from adults due to different microenvironmental influences rather than differences in dendritic cell maturation states.     

Effect of ovarian hormones on maturation of dendritic cells from peripheral blood monocytes in dogs

Previously, we reported that ovarian hormones affect the immune response against E. coli isolated from the dogs affected with pyometra. In order to investigate mechanisms underlying the immune modulation, we examined the effects of ovarian hormones on the generation of dendritic cells (DCs), the most potent antigen presenting cell. DCs were differentiated from peripheral blood monocytes (PBMOs) using a cytokine cocktail. Both estrogen receptor and progesterone receptors were expressed by the PBMOs and immature DCs. When various ovarian hormones were added to the culture for the DC differentiation, progesterone significantly decreased the expression of DC maturation markers, such as CD1a, CD80 and CD86, on mature DCs. Conversely, the addition of estrogen to the cultures increased the expression of CD86, but not other maturation makers. Furthermore, DCs differentiated in the presence of progesterone did not stimulate allogeneic mononuclear cells in PB. Taken together, these results indicate that progesterone diminishes the maturation of DCs, leading to decreased immune responses against invading pathogens.     

Human intravenous immunoglobulin (hIVIG) inhibits anti-CD32 antibody binding to canine DH82 cells and canine monocytes in vitro

The IgG receptors CD16 and CD32 (Fc_γRIII and Fc_γRII) link the humoral immune response to effector cell immune responses by binding immune complexes. Human intravenous immunoglobulin (hIVIG) consisting of immunoglobulin from pooled donors is reported to block Fc_γRs and has been used to treat a variety of canine autoimmune disorders. Fc_γRs have been poorly described for canine monocytes; therefore, the objectives of this study were to: (1) identify canine monocyte/macrophage Fc_γR (CD16 and CD32) expression and (2) demonstrate in vitro hIVIG binding to these receptors. The canine monocyte/macrophage-like cell line (DH82) and monocytes isolated from peripheral blood of healthy dogs were evaluated by flow cytometry (FACS) for CD16 and CD32 expression using commercially available anti-CD16 and anti-CD32 antibodies directed against the human isoforms. The mean percentage of cells expressing CD16 was 55% of DH82 cells and 13% of blood monocytes and the mean percentage of cells expressing CD32 was 85% of DH82 cells and 73% of blood monocytes. Immunoprecipitation of canine DH82 cells lysate using the same anti-CD16 or anti-CD32 antibodies suggested that these anti-human antibodies recognize the canine homologues. To demonstrate Fc_γR blockade, cells were incubated with increasing concentrations of hIVIG and then incubated with anti-CD16 or anti-CD32 antibodies. The percentage of CD32 expression decreased in a concentration dependent fashion in DH82 cells and blood monocytes after incubation with increasing concentrations of IVIG, suggesting that hIVIG was binding to CD32 and inhibiting anti-CD32 antibody binding. The same results were not demonstrated with anti-CD16 antibody. We believe this is the first report to demonstrate Fc_γ receptors CD16 and CD32 expression on canine monocytes and in vitro CD32 binding by human IgG, which may represent one of the immunomodulatory mechanisms of hIVIG.     

New understanding of immunological mechanisms

The understanding and importance of antigen-specific immune responses after vaccination has completely changed in recent years. In the past, the focus for monitoring a vaccine-specific immune reaction was principally on the humoral branch of the immune system. The efficacy of vaccines, as assessed by the induction of protective immunity was mainly correlated with antibodies and antibody-titers. However, this correlation often failed and other parts of the immune system had also to be considered: namely, the innate immune system and the cellular branch of the antigen-specific immune system. With regard to vaccines, the innate immune system plays its main role in the effective activation of the antigen-specific immune response, in antigen-uptake and antigen-presentation. The dendritic cells (DCs) are the most important antigen presenting cells which present processed protein antigens (peptides) through MHC-molecules: MHC-class I, for the presentation of endogenous synthesised antigen; MHC-class II for exogenous antigen. Activation of DC leads to an enhanced production of cytokines and chemokines, to an up-regulation of co-stimulatory and activation molecules and also molecules for cell-cell interactions, e.g. interactions with cells of the antigen-specific immune system. T lymphocytes are the effector cells of the cellular branch of the antigen-specific immune system. They act either as MHC-class I-restricted cytolytic T lymphocytes (CTL) or as MHC-class II-restricted T-helper cells providing support for B lymphocytes (T(H)2) and the cellular part of the antigen-specific immune system (T(H)1). In order to achieve effective vaccination, the activation of all T-cell subpopulations is of advantage, but more important is the generation of antigen-specific memory T and B lymphocytes. In addition to these 'generic' immunological factors which are essential for the design of more efficacious vaccines, our detailed knowledge about feline and canine immune reactions after vaccination, which is still poor, has to be improved.     

Cell-based cancer gene therapy: breaking tolerance or inducing autoimmunity?

This review examines the mechanisms involved in anti-tumor immunity and how peptides present in many tumor types (tumor-associated antigens) are recognized by T cells from tumor-bearing cancer patients. Tumor-associated antigens are derived from proteins that are also expressed in normal cells. It is predicted that immune responses to such peptides will be compromised by self-tolerance or that stimulation of effective immune responses will be accompanied by autoimmunity. We also consider that the immunity induced against two autoantigens, which are highly conserved in vertebrates, involve qualitatively different mechanisms, such as the production of antibodies and cell-mediated immune responses. However, both pathways lead to tumor immunity and identical phenotypic manifestations of autoimmunity. Appropriate selection of the optimal tumor antigen is critical for the induction of an anti-tumor immune response. Thus, we stress that the methods for antigen presentation using dendritic cells play a critical role in the development of tumor vaccines, to break immune tolerance and induce a strong immune response against them. The viability and feasibility of expansion of canine dendritic cells from bone marrow and peripheral blood ex vivo for the treatment of spontaneous cancers in dogs is also discussed.     

Expression and function of Toll-like receptors on dendritic cells and other antigen presenting cells from non-human primates

Antigen presenting cells (APCs), especially dendritic cells (DCs), play a crucial role in immune responses against infections by sensing microbial invasion through Toll-like receptors (TLRs). In this regard, TLR ligands are attractive candidates for use in humans and animal models as vaccine adjuvants. So far, no studies have been performed on TLR expression in non-human primates such as rhesus macaques. Therefore, we studied the TLR expression patterns in different subsets of APC in rhesus macaques and compared them to similar APC subsets in human. Also, expression was compared with corresponding DC subsets from different organs from mice. Here we show by semi-quantitative RT-PCR, that blood DC subsets of rhesus macaque expressed the same sets of TLRs as those of human but substantially differed from mouse DC subsets. Macaque myeloid DCs (MDCs) expressed TLR3, 4, 7 and 8 whereas macaque plasmacytoid DCs (PDCs) expressed only TLR7 and 9. Additionally, TLR expression patterns in macaque monocyte-derived dendritic cells (mo-DCs) (i.e., TLR3, 4, 8 and 9), monocytes (i.e., TLR4, 7, and 8) and B cells (i.e., TLR4, 7, 8, and 9) were also similar to their human counterparts. However, the responsiveness of macaque APCs to certain TLR ligands partially differed from that of human in terms of phenotype differentiation and cytokine production. Strikingly, in contrast to human mo-DCs, no IL-12p70 production was observed when macaque mo-DCs were stimulated with TLR ligands. In addition, CD40 and CD86 phenotypic responses to TLR8 ligand (poly U) in mo-DCs of macaque were higher than that of human. Despite these functional differences, our results provide important information for a rational design of animal models in evaluating TLR ligands as adjuvant in vivo.     

Development and characterization of a monoclonal antibody against porcine CD205

The aim of this work was to develop mAbs against porcine CD205 and to conduct a comparative analysis of the CD205 protein expression on lymphoid tissues, monocyte-derived dendritic cells (DCs) and DCs isolated from the porcine skin. To conduct this study, we generated a monoclonal antibody, designated 1.F6F6, against the C-type lectin-like domain-5 of the porcine CD205 and showed that it recognizes a protein band of ～200 kDa by Western Blot analysis in mesenteric lymph nodes cells. Flow cytometric analysis showed that the mAb 1.F6F6 recognized 28.5%, 28.1% and 34.1% of cells from tonsil, inguinal and mesenteric lymph nodes, respectively, and 6% of cells from thymus. Analysis of monocyte-derived DCs showed that approximately 20% were positive and activation of the cells with LPS increased the positive population to 36%. Analysis of DCs isolated from the porcine skin showed that approximately 70% of the cell population expressed the CD205 receptor. The development of a monoclonal antibody capable of recognizing the CD205 receptor in swine opens up possibilities of applying new strategies for enhancing vaccine efficacy by using the anti-CD205 antibody for DC antigen-targeting to enhance priming of immune responses.     

CD20 expression in normal canine B cells and in canine non-Hodgkin lymphoma

We examined the expression of CD20 in normal canine peripheral blood mononuclear cells, normal canine spleen, and canine non-Hodgkin lymphoma (NHL) to determine the feasibility of using this antigen as a diagnostic aid and as a possible target for therapy. An antibody generated against a C-terminal (intracytoplasmic) epitope of human CD20 recognized proteins of 32-36 kd in normal and malignant canine lymphocytes. This antibody showed restricted membrane binding in a subset of lymphocytes in peripheral blood, in the B-cell regions from a normal canine spleen and lymph node, and in malignant cells from 19 dogs with B-cell NHL, but not from 15 dogs with T-cell NHL. The patterns of CD20 reactivity in these samples overlapped those seen using an antibody that recognizes canine CD79a. This anti-CD20 antibody is therefore suitable as an aid to phenotype canine NHL. In contrast, normal canine B cells were not recognized by any of 28 antibodies directed against the extracellular domains of human CD20 (including the chimeric mouse-human antibody Rituximab) or by any of 12 antibodies directed against the extracellular domains of mouse CD20. Thus, the use of CD20 as a therapeutic target will require the generation of specific antibodies against the extracellular domains of canine CD20.     

Characterization of antibodies specific for canine TLR4, 5 and 9 by ELISA, Western blotting and immunohistochemistry

Toll-like receptors recognize pathogen-associated molecular patterns of microbial origin, and ligand recognition results in the production of different immune mediators such as pro-inflammatory cytokines, interferon, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. As these receptors have a critical role in linking pathogen recognition to induction of inflammation and innate as well as adaptive immunity, there is tremendous interest in understanding how the tissue and cell-type expression of TLRs is regulated and its influence on the local innate immune response. While TLRs are well studied in humans and rodents, to date little is known about them in dogs. The purpose of this study was to develop canine specific antibodies against TLR2, 4, 5 and 9 that were used to measure relative expression of these TLRs in healthy and reactive canine mesenteric lymph nodes. All 8 rabbit sera (2 each for TLR2, 4, 5 and 9) were strongly positive in ELISA against the respective 2 peptides per TLR used for immunization. The purified antibodies selected specifically detected a protein band with an apparent size of approximately 70 kDa in lysates of canine PBMCs by Western blotting. Immunostaining was observed with purified antibodies against TLR4, 5 and 9, whereas for canine TLR2, staining was only observed with the unpurified antibodies. In the mesenteric lymph node of healthy dogs, the overall staining pattern was very similar for TLR4 and 5 with positive cells predominantly found in the internodular areas and lower part of the cortex. Compared to the TLR4 and 5, more cells stained positive for TLR9 especially in the lymphoid nodules. The reactive lymph nodes contained more TLR4 and 9 positive cells. Moreover, a shift of TLR-9 positive cells from the lymphoid follicles to the deep cortex and medullary cords was observed. Whereas TLR9 co-localized with CD79-positive areas, TLR4 and 5 antibodies stained cells primarily in the CD3-positive areas. All three TLR antibodies stained cells within the area that co-localized with lysozyme-positive cells. In conclusion, this study demonstrates that the antibodies generated against canine TLR 4, 5 and 9 identify the expression of these TLRs in formalin-fixed canine lymph nodes and demonstrate increased expression in reactive canine mesenteric lymph nodes.     

Cell-mediated immunity and age at vaccination associated with measles inoculation and protection of dogs against canine distemper.

The antibody-mediated immune response (AMIR) of dogs to measles and canine distemper viruses has been described. However, there is little information on the cell-mediated immune response (CMIR). The AMIR and the CMIR of dogs to canine distemper and to measles were examined. The CMIR was determined for 6 weeks by measuring the 3H-thymidine uptake by immune lymphocytes in the lymphocyte transformation test. Concurrently, canine distemper and measles virus serum-neutralization antibodies were measured by a microtitration serum-neutralization test. Dogs vaccinated with canine distemper virus had a CMIR and an AMIR to canine distemper. However, measles virus-vaccinated dogs had only a CMIR to canine distemper. A CMIR in the absence of an AMIR indicates that cell-mediated immunity is the most important immune mechanism in protecting measles virus-vaccinated dogs against canine distemper. Development of CMIR and AMIR to canine distemper and measles antigens depended on the age of the dog at the time of vaccination. Adult and juvenile dogs had immune responses to both canine distemper and measles. Neither virus, however, elicited an immune response in neonates.     

Engineered cross‐reacting nanobodies simplify comparative oncology between humans and dogs

Antibodies cross‐reacting with homologue antigens in different species would be essential reagents for the development of comparative oncology studies. In comparison with conventional immunoglobulin Gs, recombinant nanobodies (single‐domain variable regions of heavy‐chain only antibodies of Camelidae origin) can be easily isolated in vitro and engineered into a variety of reagents with optimized characteristics for different research and clinical applications. We recovered an anti‐human epidermal growth factor receptor 2 (anti‐HER2) nanobody from a naïve llama library by direct panning on whole cells and expressed it fused to Fc and green fluorescent protein. These immunoreagents were assessed by flow cytometry and immunofluorescence with both human and canine cells overexpressing HER2 and its canine homologue dog epidermal growth factor receptor 2. The reported data illustrate the potential of using this class of antibodies in comparative oncology and suggest some development perspectives enabled by in vitro panning of pre‐immune nanobody libraries.     

Induction of dendritic cell-mediated immune responses against canine malignant melanoma cells

To establish the basis for the use of dendritic cells (DC) in the treatment of canine melanoma, dogs were vaccinated using autologous DC pulsed with canine melanoma CMM2 cell lysate in the presence of keyhole limpet haemocyanin (KLH) in vitro (CMM2-KLH-DC), and the induction of immune responses against CMM2 cells in vivo was examined using the delayed-type hypersensitivity (DTH) skin test. The DTH responses against CMM2 cells and KLH were observed in dogs vaccinated with CMM2-KLH-DC, while the responses against KLH but not CMM2 cells were detected with DC pulsed with KLH alone (KLH-DC). Recruitment of CD8 and CD4 T cells was detected in the positively responding sites, suggested that vaccination with CMM2-KLH-DC efficiently elicits T cell-mediated immunity against CMM-2 cells in vivo. These findings demonstrate the potential utility of DC-based tumour vaccination in the treatment of canine malignant melanoma.     

Comparative immunology

As a discipline, comparative immunology enhances zoology and has gained wide acceptance in the biological sciences. It is an offshoot of the parent field, immunology, and is an amalgam of immunology and zoology. All animals from protozoans to humans have solved the threat of extinction by having evolved an immune‐defense strategy that ensures the capacity to react against foreign, non‐self microorganisms and cancers that disturb the homeostatic self. Invertebrate‐type innate immune responses evolved first and they characterize the metazoans. These rapid natural responses act immediately and are often essential for the occurrence of slower, more specific, adaptive vertebrate‐type immune responses. As components of the innate immune system, there is an emphasis on several major steps in the evolutionary process: (i) recognition; (ii) the phagocytic cell; and (iii) the natural killer cell. When vertebrates evolved, beginning with fish, thymus‐controlled T cells first appeared, as did bone marrow‐derived B cells (first found in amphibians with long bones). These were the precursors of the plasma cells that synthesize and secrete antibodies. Confirming the concept of self/non‐self, invertebrates possess natural, non‐adaptive, innate, non‐clonal, non‐anticipatory immune responses, whereas vertebrates possess adaptive, acquired, clonal, and anticipatory responses. This symposium concerns: (i) aspects of the immune spectrum in representative groups; (ii) specific findings (in particular models; e.g. earthworms); (iii) clues as to the possible biomedical application of relevant molecules derived from animals, notably invertebrates; and (iv) some views on the more practical applications of understanding immune systems of invertebrates and ectotherms, and their possible role in survival.     

The cytoskeletal intermediate filaments of canine retinal pigment epithelial cells in vivo and in vitro

The cytoskeletal intermediate filament characteristics of normal, freshly isolated and subcultured canine retinal pigment epithelial ( ) cells were studied using immunocytochemistry and immunoblotting techniques. Commercially available primary antibodies recognising a broad range of cytokeratins and vimentin were selected. Cytokeratin reactivity was a constant feature of all canine cells. The main cytokeratins expressed by cultured RPE cells included 8, 18 and 19. This finding is consistent with the published findings of work carried out in other mammalian species including man. Freshly isolated cells stained positively with broad-spectrum antibodies to cytokeratins but generally did not stain with antibodies specific to cytokeratins 18 or 19 and did not stain with antibodies to vimentin, or stained only very weakly. After a short time in culture however, cells demonstrated intense positive staining for vimentin. This study demonstrated that cytokeratin immunoreactivity (in conjunction with vimentin immunoreactivity in vitro) is a useful and consistent marker for canine RPE cells.     

Characterization of monoclonal antibodies against canine P-selectin glycoprotein ligand-1 (PSGL-1)

Thirteen different monoclonal antibodies against canine P-selectin glycoprotein ligand-1 (cPSGL-1) were obtained by immunization of rats with cells of a canine lymphoma cell line (Ema). O-sialoglycoprotein endopeptidase treatment of Ema cells showed that all of these antibodies recognized O-glycosylated peptides of canine PSGL-1. Experiments using deletion or point mutants of cPSGL-1 indicated that these antibodies could be categorized into several groups based on their cPSGL-1 recognition characteristics. These anti-cPSGL-1 monoclonal antibodies will be useful for analysis of the canine P-selectin and PSGL-1 system.     

Display and selection of chicken IgA Fab fragments

Passive immune therapy is regaining interest to prevent and cure infectious diseases both in human and veterinary medicine. Therefore, systems are required that enable efficient targeted selection of antibodies originating from virtually any animal species. Here, a system for the selection of chicken IgA, using phage display, is described. A novel phagemid vector (pChick3) for the display and selection of chicken IgA antibodies in Fab format was developed. The functionality of pChick3 was demonstrated by construction of an immune antibody library using B cells from chickens infected with Eimeria acervulina. From this library, 10 different IgA fragments with specific binding to the E. acervulina antigen mix, the sporozoite or oocyst fractions were selected. These results demonstrate the efficiency and versatility of the pChick3 vector system that can readily be applied to construct libraries and subsequently select antibodies of the alpha isotype against a wide variety of pathogens and parasites.     

Cross-reactive anti-human monoclonal antibodies as a tool for B-cell identification in dogs and pigs

We have characterized a panel of commercially available anti-human monoclonal antibodies (mAbs) suitable for B-cell identification in pigs and dogs. The specificities of the mAbs were against CD20, CD21, CD22, and CD86. In addition to HM57, originally raised against human CD79alpha the broad cross-reactivity of which was documented more than 10 years ago, we recommend here a panel of several other mAbs as a useful tool for immunophenotyping and multicolor flow cytometry of canine and porcine B-lymphocytes. All six investigated antibodies did bind weakly to either canine or porcine lymphocytes (or both), but considerable weaker than for the human control cells. Four of them did bind to canine or porcine spleen section in immunohistochemistry. Monoclonal antibody against CD22 (clone RFB-4) was the only antibody in the tested panel the cross-reactivity of which was confirmed by Western blot. The advantages and limits of cross-reactive mAbs in studies on animal B-cells are discussed.