Clinical features of canine nodal T‐cell lymphomas classified as CD8+ or CD4−CD8− by flow cytometry

Canine T‐cell lymphoma (TCL) encompasses a heterogeneous group of diseases with variable clinical presentation, cytomorphology, immunophenotype, and biologic behaviour. The most common types of TCL in dogs involving peripheral lymph nodes include indolent T‐zone lymphoma (TZL) and biologically aggressive peripheral T‐cell lymphoma (PTCL). TCL phenotypes can be categorized by expression of the surface antigen molecules CD4 and CD8. The majority of TCL cases are CD4⁺, with far fewer cases being CD8⁺ or CD4^? CD8^?. The clinical features of CD4⁺ TCLs have been previously described. The less common TCL phenotypes, however, are poorly characterized with little to no information about prognosis. In this retrospective study, we describe and correlate the presenting clinical signs, flow cytometry, and outcomes of 119 dogs diagnosed with nodal, non‐TZL, CD8⁺ or CD4^? CD8^? TCL by flow cytometry. Skin lesions present at the time of diagnosis were more commonly observed in the CD8⁺ TCL group. Mediastinal enlargement and/or hypercalcemia were more commonly seen in the CD4^? CD8^? TCL group. Dogs with either CD8⁺ or CD4^? CD8^? TCLs had aggressive clinical disease with median overall survival (OS) times of 198 days and 145 days, respectively. In both groups, neoplastic cell size determined by flow cytometry ranged from small to large, and large cell size was associated with shorter OS times (median OS = 61 days). Cases classified as small cell had a median OS of 257 days. Expression levels of major histocompatibility complex (MHC) class II and CD5 were highly variable among cases but were not prognostically significant in this group of patients.     

Canine CD8 T cells showing NK cytotoxic activity express mRNAs for NK cell-associated surface molecules

Natural killer (NK) cells have been considered to be a group of lymphocytes lacking clonally distributed receptors for antigens typical of T cells and B cells. In some mammalian species, including humans, a subpopulation of CD8⁺ peripheral blood lymphocytes (PBLs) exhibits NK activity. This NK subpopulation has not been well characterized in mammals and its characterization is particularly poor in the dog. In this study, we demonstrated that a subset of canine CD8⁺ cells derived from PBLs and lymphokine (IL-2)-activated killers (LAKs) of PBLs that was CD3⁺, CD4^?, CD21^?, CD5^lo, α/βTCR⁺, and γ/δTCR^? contained substantially higher levels of mRNAs for NK cell-related receptors (NKp30, NKp44, NKG2D, 2B4, and CD16 for PBL, and NKG2D and CD56 for LAK) than the corresponding CD8^? cells. This subset of CD8⁺ lymphocytes derived from LAKs also displayed significantly higher NK cytotoxic activity than the corresponding CD8^? cells. In contrast, CD8⁺ cells derived from nonstimulated PBLs showed very low levels of NK cytotoxic activity. Our results indicate that, in IL-2-stimulated PBLs, canine CD8⁺ cells are an important subset associated with NK cytotoxic activity.     

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.     

Lymphocyte Populations in Joint Tissues from Dogs with Inflammatory Stifle Arthritis and Associated Degenerative Cranial Cruciate Ligament Rupture

Objective: To evaluate lymphocyte populations in stifle synovium and synovial fluid of dogs with degenerative cranial cruciate ligament rupture (CCLR). Study Design: Prospective clinical study. Animals: Dogs (n=25) with stifle arthritis and CCLR, 7 dogs with arthritis associated with cartilage degeneration (osteoarthritis [OA]), and 12 healthy Beagle dogs with intact CCL. Methods: Arthritis was graded radiographically in CCLR dogs. After collection of joint tissues, mononuclear cells were isolated and subsequently analyzed using flow cytometry for expression of CD3, CD4, CD8, and CD21. Results: The proportions of CD4⁺ T helper lymphocytes, CD8⁺ cytotoxic T lymphocytes, and CD3⁺CD4^?CD8^? T lymphocytes were increased in synovium from dogs with CCLR compared with synovium from healthy Beagle dogs (P<.05). The proportion of CD3⁺CD4^?CD8^? T lymphocytes in synovial fluid was increased in dogs with CCLR compared with dogs with OA (P<.05). In dogs with CCLR, the proportion of CD3⁺CD4^?CD8^? T lymphocytes in synovial fluid was inversely correlated with radiographic arthritis (S_R=?0.68, P<.005). Conclusion: Lymphocytic inflammation of stifle synovium and synovial fluid is an important feature of the CCLR arthropathy. Lymphocyte populations include T lymphocytes expressing CD4 and CD8, and CD3⁺CD4^?CD8^? T lymphocytes. Presence of CD3⁺CD4^?CD8^? T lymphocytes was associated with development of stifle synovitis. Further work is needed to fully identify the phenotype of these cells.     

Cutaneous T‐cell lymphoma – Sézary syndrome in a Boxer

A 7‐year‐old male Boxer with a 3.5‐year history of atopy and food hypersensitivity was presented with multiple poorly circumscribed nodules and maculae of the skin and tongue, and jaundiced mucosal membranes. Cytologic and histopathologic examination of the skin lesions revealed cutaneous epitheliotropic lymphoma. Cells were CD3⁺ and CD8⁺ in flow cytometry. The CBC showed a moderate leukocytosis with 16% atypical lymphocytes with irregularly cleaved nuclei. Flow cytometric phenotyping of peripheral blood showed an elevated proportion of the CD8⁺ T‐lymphocyte subpopulation, indicating a malignant population of T‐cell origin, and the electropherogram of the PCR antigen receptor rearrangement produced a monoclonal peak for TCRγ. Liver enzyme activities were markedly increased and abdominal ultrasound examination showed increased echogenicity of the liver and enlarged abdominal lymph nodes. Fine‐needle aspirates of the liver confirmed infiltration with lymphocytes exhibiting the same morphology as the cells detected in skin and peripheral blood. Treatment was induced with L‐asparaginase, lomustine, and prednisone. Partial clinical remission of the skin and tongue lesions was achieved within 10 days, and hematologic abnormalities resolved. Despite further treatment with L‐asparaginase and lomustine, the dog relapsed within one month and was euthanized. Presence of malignant lymphocytes in skin, peripheral blood, and liver indicate a rare variant of leukemic cutaneous T‐cell lymphoma, equivalent of Sézary syndrome in a dog. This case report describes the use of flow cytometry as a complementary tool for lymphocyte characterization of skin lesions for the first time.     

Assessing the immune state of dogs suffering from pituitary gland dependent hyperadrenocorticism by determining changes in peripheral lymphocyte subsets

In order to evaluate the immune state of dogs suffering from pituitary-dependent hyperadrenocorticism (PDH), peripheral lymphocyte subsets were examined. Twenty seven PDH dogs and eight healthy control dogs were used in the current study. Eight healthy dogs served as the control group. Twenty seven PDH dogs were categorized into 4 groups based on their post serum cortisol concentrations by ACTH stimulation test: 2−5, excellent control (n = 8); 5−20, fair control (n = 7); >20, poor control (n = 4); and untreated (n = 8). Cell counts were executed with white blood cells (WBC), lymphocytes, CD3⁺ (T lymphocytes), CD4⁺ (Helper T lymphocytes), CD8⁺ (Cytotoxic T lymphocytes), CD21⁺ (B lymphocytes) cells in addition to calculating CD4⁺/CD8⁺ ratio. Results indicated a significant difference in lymphocyte numbers and lymphocyte subset populations (CD3⁺, CD4⁺, CD8⁺, and CD21⁺ cells) between PDH and control dogs. Moreover, comparison of the PDH groups (excellent control; fair control; poor control; untreated) demonstrated that all groups had a significant decrease in lymphocytes numbers (CD3⁺, CD4⁺ and CD21⁺ cell counts) as compared to control group. Meanwhile, no significant differences were observed in WBC counts and CD4⁺/CD8⁺ ratio between groups. Furthermore, lymphocyte subset distribution in excellent control PDH dogs without concurrent disease (n = 4) better resembled that of control dogs as compared to PDH dogs with concurrent disease (n = 4). PDH dogs may be suffering from an immuno-depressed state as evidenced by significant differences in lymphocyte subset populations. Furthermore, treatment of both PDH and concurrent disease might improve lymphocyte subset distribution.     

Canine leishmaniasis transmission: higher infectivity amongst naturally infected dogs to sand flies is associated with lower proportions of T helper cells

The dog is the main reservoir of Leishmania infantum, which is a parasite spread among canine hosts by the bite of sand flies. Phlebotomus perniciosus is the sand fly acting as a major vector in the Mediterranean basin. As a consequence, the dog will suffer from leishmaniasis. In this work the infective capacity of infected dogs, established by direct xenodiagnosis, has been investigated in relation to their immunological status by determining the lymphocyte percentages present in peripheral blood mononuclear cells. We found a significant association between the percentages of T helper cells (CD4/TcRαβ⁺and CD4/CD45RA⁺) and the infection rates detected in the vector, while significant association was not detected in the case of the T cytotoxic cells (CD8/TcRαβ⁺and CD8/CD45RA⁺). The relationship discovered was that the lower the CD4⁺T cell count, the higher the rate of the infection in the vector.     

Cattle Infected with Bovine Leukaemia Virus may not only Develop Persistent B‐cell Lymphocytosis but also Persistent B‐cell Lymphopenia

We investigated the distribution of B and T cells in the peripheral blood of haematologically inconspicuous (non‐persistent lymphocytotic, PL^?) cattle infected with the bovine leukaemia virus (BLV). Flow cytometric data were obtained from six PL^? cattle and compared with six age‐matched animals with persistent lymphocytosis (PL⁺) and five non‐infected healthy controls (BLV^?). In the PL^? group, the percentage and number of surface immunoglobulin‐positive (sIg⁺) B cells were significantly reduced. Whereas in BLV^? cattle, about 40% of the peripheral blood lymphocytes (PBL) were sIg⁺ and 24% were sIgM⁺ B cells. In the PL^? group, less than 20% of the PBL were sIg⁺ and sIgM⁺ B cells. Only 5% of the PBL co‐expressed sIgM⁺ and CD5⁺ versus 16% in BLV^?. This decrease was persistent over 3 years and predominantly affected: (i) B cells that did not express sIgM; (ii) sIgM⁺ B cells co‐expressing CD5 and CD11b; and (iii) equally both λ‐ and κ‐type light chain B‐cell subpopulations. In contrast, the number of all circulating lymphocytes, CD5^? and CD11b^? sIgM⁺ B cells and CD2⁺ T cells did not differ. In PL⁺ animals, about 75% of the PBL were sIgM⁺CD5⁺ B cells. These cells were of polyclonal origin, as light chains of the λ‐ and κ‐type were expressed in a ratio of 4:1 (57.7% of PBL λ⁺, 14% κ⁺) as in BLV^? animals (33.6% of PBL λ⁺, 8.7% κ⁺). In PL⁺ cattle the absolute number of B‐cells and, therefore, their relative percentage is significantly increased. For this reason, even in case of absolutely increased T‐cell numbers, the relative percentage of T‐cells could be lower than in normal controls. The cause for the observed B cell decrease in PL^? cattle is unknown, but it can be assumed that cytotoxic T cells are involved in this B‐cell lymphopenia.     

Unique features of bovine lymphocytes exposed to a staphylococcal enterotoxin

We previously demonstrated that stimulation of bovine peripheral blood mononuclear cells (PBMCs) with staphylococcal enterotoxin C (SEC), led to an inversion of the CD4⁺:CD8⁺ T cell ratio and generation of an atypical CD8⁺ T cell subpopulation expressing CD26. In the present study, we examined T cell apoptosis and proliferation profiles of PBMC subpopulations in cultures stimulated with SEC. Unlike when stimulated with concanavalin A, nucleic acid synthesis in bovine PBMC cultures stimulated with SEC was low during the first four days but increased greatly on day 5. In contrast, nucleic acid synthesis in human PBMC cultures stimulated with SEC increased continuously. To investigate the mechanism of delayed bovine T cell proliferation, various cell phenotypes were monitored. The inversion of the bovine CD4⁺:CD8⁺ T cell ratio in PBMC cultures stimulated by SEC was associated with higher proliferation and lower apoptosis of CD8⁺ T cells compared to CD4⁺ T cells. The mRNA levels for interleukin (IL)-4 and IL-13 were sustained over 4 days but IL-12 mRNA levels dropped to background on day 2. These data suggest that SEC induces a prolonged Th-2-biased microenvironment, and together with the inversion of the bovine CD4⁺:CD8⁺ T cell ratios in bovine PBMC cultures with SEC, may in part explain the inability of the mammary immune system to establish an effective response to Staphylococcus aureus infections.     

Studies on the immune status of calves with chronic inflammation and thymus atrophy

The thymus is a primary lymphoid organ where the primary T cell repertoire is generated. Thymus atrophy is induced by various conditions, including infectious diseases, glucocorticoid treatment, and poor breeding management. Cattle with thymus atrophy tend to exhibit weak calf syndrome, a condition in which approximately half of neonates die shortly after birth. Calves with thymus atrophy that survive the first month typically contract chronic inflammatory diseases. In this study, we analyzed the populations of the peripheral blood mononuclear cells and thymocytes in calves with thymus atrophy. In addition, we evaluated polarization of master gene and cytokine mRNA expression in peripheral blood CD4⁺ cells in the calves. The population of CD4⁺CD8⁺ cells in thymus of the calves with thymus atrophy was lower than that of control calves. IL10 mRNA expression in peripheral blood CD4⁺ cells of calves with thymus atrophy was significantly lower than that of control calves. TBX21 mRNA expression in peripheral CD4⁺ cells of thymus atrophy calves was tended to be higher than that of the control group. In addition, FOXP3 mRNA expression in peripheral CD4⁺ cells of the thymus atrophy calves was tended to be lower than that of the control calves. Thymus atrophy calves exhibited chronic inflammatory disease leading, in severe situations, to conditions such as pneumonia with caseous necrosis. These severe inflammatory responses likely are due to decreases in IL10 mRNA expression, impairing control of macrophages, one of the main cell fractions of natural immunity.     

An engineered anti-idiotypic antibody-derived killer peptide (KP) early activates swine inflammatory monocytes,CD3+CD16+ natural killer T cells and CD4+CD8α+ double positive CD8β+ cytotoxic T lymphocytes associated with TNF-α and IFN-γ secretion

This study evaluated the early modulation of the phenotype and cytokine secretion in swine immune cells treated with an engineered killer peptide (KP) based on an anti-idiotypic antibody functionally mimicking a yeast killer toxin. The influence of KP on specific immunity was investigated using porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) as ex vivo antigens. Peripheral blood mononuclear cells (PBMC) from healthy pigs were stimulated with KP and with a scramble peptide for 20 min, 1, 4 and 20 h or kept unstimulated. The cells were analyzed using flow cytometry and ELISA. The same time-periods were used for KP pre-incubation/co-incubation to determine the effect on virus-recalled interferon-gamma (IFN-γ) secreting cell (SC) frequencies and single cell IFN-γ productivity using ELISPOT.KP induced an early dose-dependent shift to pro-inflammatory CD172α⁺CD14^+high monocytes and an increase of CD3⁺CD16⁺ natural killer (NK) T cells. KP triggered CD8α and CD8β expression on classical CD4⁻CD8αβ⁺ cytotoxic T lymphocytes (CTL) and double positive (DP) CD4⁺CD8α⁺ Th memory cells (CD4⁺CD8α^+low CD8β^+low). A fraction of DP cells also expressed high levels of CD8α. The two identified DP CD4⁺CD8α^+high CD8β^+low/+high CTL subsets were associated with tumor necrosis factor alpha (TNF-α) and IFN-γ secretion. KP markedly boosted the reactivity and cross-reactivity of PRRSV type-1- and PCV2b-specific IFN-γ SC. The results indicate the efficacy of KP in stimulating Th1-biased immunomodulation and support studies of KP as an immunomodulator or vaccine adjuvant.     

Porcine reproductive and respiratory syndrome virus induces pronounced immune modulatory responses at mucosal tissues in the parental vaccine strain VR2332 infected pigs

Porcine reproductive and respiratory syndrome (PRRS) is a chronic viral disease of pigs caused by PRRS virus (PRRSV). The PRRSV VR2332 is the prototype North American parental strain commonly used in the preparation of vaccines. Goal of this study was to understand missing information on VR2332 induced immune modulation at the lungs and lymphoid tissues, the sites of PRRSV replication. Pigs were infected intranasally and samples collected at post-infection day (PID) 15, 30, and 60. Microscopically, lungs had moderate interstitial pneumonia, and the virus was detected in all the tested tissues. Peak antibody response and the cytokine IFN-γ secretion were detected at PID 30, with increased TGF-β until PID 60. Population of CD8⁺, CD4⁺, and CD4⁺CD8⁺T cells, Natural killer (NK) cells, and γδ T cells in the lungs and lymphoid tissues were significantly modulated favoring PRRSV persistence. The NK cell-mediated cytotoxicity was significantly reduced in infected pigs. In addition, increased population of immunosuppressive T-regulatory cells (Tregs) and associated cytokines were also observed in VR2332 strain infected pigs.     

Effect of dexamethasone and meloxicam on counts of selected T lymphocyte subpopulations and NK cells in cattle – In vivo investigations

The purpose of these investigations has been to assess the in vivo effect of dexamethasone (DEX) and meloxicam (MEL) on percentages and absolute counts of cells within selected T lymphocyte subsets and NK cells in cattle. DEX application caused substantial loss of NK, CD4⁺, CD8⁺ and WC1⁺ T cells, but the drug’s influence on T-cell count was selective, that is it varied according to the presence and intensity of CD25 expression. Reduced counts of T lymphocytes were due to the depletion of CD25⁻CD4⁺, CD25⁻CD8⁺ and CD25⁻WC1⁺ T cells. The loss of CD25⁻CD8⁺ and CD25⁻WC1⁺ T cells was a deep and lasting disorder, whereas the depletion of CD25⁻CD4⁺ T cells was manifested less strongly and regressed promptly. The administration of DEX did not affect absolute counts of CD25^lowCD4⁺ and CD25^lowCD8⁺ T cells, but induced an increase in percentages and absolute counts of CD25^highCD4⁺, CD25^highCD8⁺, CD25^lowWC1⁺ and CD25^highWC1⁺ T cells. In respect of the effect on counts of CD4⁺, CD8⁺ and WC1⁺ T cells, MEL proved to be a safe medication, because it did not alter counts of these lymphocytes. The administration of MEL led to an increase in the absolute count of NK cells, but the effect did not appear quickly and its development required time.     

Lymphocytosis and lymphadenopathy in a dog arising from two distinct lymphoid neoplasms

A 10-year-old intact male Golden Retriever was presented to The Ohio State University Veterinary Medical Center for acute, non-painful facial swelling of the right mandibular region. On physical examination, the right mandibular swelling was found to represent marked lymphadenopathy of the submandibular lymph node. At this time, marked lymphadenopathy of the prescapular and popliteal lymph nodes was also appreciated. The CBC showed a moderate leukocytosis (38.4 × 10⁹ cells/L, reference interval [RI] 4.8-13.9 × 10⁹ cells/L) characterized by a moderate lymphocytosis (28.4 × 10⁹ cells/L, RI 1.0-4.6 × 10⁹ cells/L). Evaluation of peripheral blood and enlarged prescapular and popliteal lymph nodes revealed two morphologically different populations of homogeneous lymphocytes, with the lymphocyte population in the lymph nodes being distinct from that in the blood smear. Flow cytometry of peripheral blood revealed CD45-, CD5+, CD4-, CD8-, variably CD21+ neoplastic lymphocytes compatible with T-zone lymphocytes due to the absence of CD45 expression. Flow cytometry of the lymph node aspirate indicated a distinct population of CD21+ lymphocytes consistent with a B-cell phenotype along with a smaller proportion of the T-zone lymphocytes observed in the blood confirming the presence of two distinct populations of neoplastic lymphocytes, one involving T cells, and the other involving B cells.     

Factors affecting disease manifestation of toxocarosis in humans: Genetics and environment

Toxocara canis is regarded as the main cause of human toxocarosis but the relative contribution of T. cati is probably underestimated; serological and other diagnostic methods used in most studies of this zoonotic disease do not distinguish between the two parasites. The definitive hosts for T. canis are caniidae. Pups generally have higher infection rates than adult animals and are a major source of eggs in the environment. Humans usually acquire T. canis infection by accidental ingestion of embryonated eggs or encapsulated larvae from the environment or contaminated food, such infections may lead to visceral larva migrans (VLM), ocular larva migrans (OLM) or covert toxocarosis (CT). Although a mixed Th1- and Th2-mediated immunological response, particularly with high levels of IgE and eosinophilia is observed, the underlying mechanisms of molecular and immunopathogenesis for the development of the symptomatic syndromes of VLM, OLM, or of asymptomatic CT are largely unclear. Studies have indicated that immunological defences against various infectious diseases may be highly influenced by complex interactions of environmental and host genetic factors e.g. MHC class I and II, also known as human leucocyte antigen (HLA). Toxocara spp. infections are associated with a polarized CD4⁺ Th2 response with high IgE levels and eosinophilia, mediated mainly by HLA class II molecules. Associations have been made between HLA class II and pathological severity and host genetic effects on exposure to infection. Recent research suggests Foxp3⁺ CD4⁺CD25⁺-expressing T regulatory (Treg) cells play a role in regulation of the immunopathology of granulomas in experimental toxocaral granulomatous hepatitis and in enhanced expression of TGF-β1, which is an important factor for the local survival and function of Treg observed during T. canis invasion in the mouse small intestine, liver, muscle, and brain. Since the potential susceptibility loci HLA class II molecules, are considered involved in the regulation of a Th2-dominant immunity which is highly controlled by Foxp3⁺ CD4⁺CD25⁺ Treg cells by stimulation through TGF-β1, which thus provides a beneficial environment to T. canis larvae but severe injuries to local organs. However, TGF-β1 variant Leu10Pro known to be involved in disease severity warrants further elucidation as this too may have a role in the severity of human toxocarosis. Exploration of TGF-β1 polymorphism, Foxp3⁺ CD4⁺CD25⁺ Treg cells, and MHC polymorphisms may allow insight into the contribution made by environmental and genetic factors in influencing disease syndrome type and severity in humans with toxocarosis.     

CD27 expression discriminates porcine T helper cells with functionally distinct properties

Differentiation of porcine T helper cells is still poorly investigated, partly due to a lack of monoclonal antibodies (mAbs) specific for molecules involved in this process. Recently, we identified a mAb specific for porcine CD27 and showed that CD27 is expressed by all naïve CD8α^- T helper cells but divides CD8α⁺ T helper cells into a CD27⁺ and a CD27^- subset. In the present study, detailed phenotypical and functional analyses of these T-helper cell subpopulations were performed. Naïve CD8α^-CD27⁺ T helper cells predominantly resided in various lymph nodes, whereas higher proportions of CD8α⁺CD27⁺ and CD8α⁺CD27^- T helper cells were found in blood, spleen and liver. Both CD8α⁺CD27⁺ and CD8α⁺CD27^- T helper cells were capable of producing IFN-γ upon in vitro polyclonal stimulation and antigen-specific restimulation. Experiments with sorted CD8α^-CD27⁺, CD8α⁺CD27⁺ and CD8α⁺CD27^- T-helper cell subsets following polyclonal stimulation revealed the lowest proliferative response but the highest ability for IFN-γ and TNF-α production in the CD8α⁺CD27^- subset. Therefore, these cells resembled terminally differentiated effector memory cells as described in human. This was supported by analyses of CCR7 and CD62L expression. CD8α⁺CD27^- T helper cells were mostly CCR7^- and had considerably reduced CD62L mRNA levels. In contrast, expression of both homing-receptors was increased on CD8α⁺CD27⁺ T helper cells, which also had a proliferation rate similar to naïve CD8α^-CD27⁺ T helper cells and showed intermediate levels of cytokine production. Therefore, similar to human, CD8α⁺CD27⁺ T helper cells displayed a phenotype and functional properties of central memory cells.     

Comparison of T-cell subpopulations in cats naturally infected with feline leukaemia virus or feline immunodeficiency virus

T-cell subsets were studied by flow cytometry in 58 feline leukaemia virus (FeLV)-positive cats with naturally acquired FeLV infection to determine whether the changes in CD4⁺ or CD8⁺ T cell populations differed from those observed in 55 feline immunodeficiency virus (FIV)-positive cats with naturally acquired FIV infection. The sole criterion for inclusion into the study was seropositivity. Mean (SD) CD4⁺ T cell values of FeLV positive cats were decreased to 31·1 (8·0) per cent and their CD8⁺ T cell values were increased to 22·8 (6·3) per cent in comparison with uninfected control cats (37·9 [9·5] per cent CD4⁺; 15·2 [6·3] per cent CD8⁺). The CD4⁺/CD8⁺ ratio was reduced to 1·5 (0·7), compared with 3·0 (1·5) in 39 FeLv- and FIV-negative control cats. Differences from control values were significant, but there was no significant difference between CD4⁺ and CD8⁺ lymphocytes of FeLV- versus FIV-infected cats. These findings indicate that FeLv and FIV have similar effects on T lymphocyte subsets. Both retrovirus infections can induce immunodeficiency, both viruses infect a broad range of lymphohaemopoietic cells, despite having different primary target cells, and can induce the killing of lymphocytic cells in vitro. It is concluded that a decreased CD4⁺/CD8⁺ ratio is not restricted to FIV infections but may also occur in FeLv infection.     

Diffuse leptomeningeal histiocytic sarcoma in the cerebrospinal fluid of 2 dogs

Two adult male castrated dogs were evaluated for progressive paraparesis and ataxia. Neurologic examination showed severe ataxia, delayed proprioceptive placement in the pelvic limbs, pain upon palpation of the lumbar spine as well as facial paresis in one dog, and decreased withdrawal reflex of the pelvic limbs in the other dog. Magnetic resonance imaging (MRI) in both dogs showed diffuse meningeal and intramedullary lesions. However, no evidence of a mass was found. Biopsies could not be performed safely due to the location of the lesions. Cerebrospinal fluid (CSF) examination revealed an inflammatory pleocytosis associated with increased protein concentration and numerous large atypical round cells, often multinucleated. Nuclear fragmentation, micronuclei, and rare atypical mitoses were observed. Immunocytochemistry revealed CD1⁺ and CD11c⁺ staining, which, in concert with the morphology confirmed the diagnosis of histiocytic sarcoma (HS). Euthanasia was elected due to poor prognosis. Histopathologic examination showed diffuse spinal and meningeal infiltration with CD18⁺ neoplastic cells, without any evidence of mass formation, which completed the diagnosis of diffuse leptomeningeal HS involving the brain and the spinal cord. Canine central nervous system (CNS) HS has been seldom reported in the literature, with only isolated cases identified on CSF cytology. The cases reported here are remarkable in describing a diffuse CNS leptomeningeal HS associated with neoplastic cells in the CSF of dogs without a tumor mass. These cases emphasize the potential critical importance of CSF analysis in providing an antemortem diagnosis of neoplasia in neurologic patients.     

Postnatal Development of Lymphocyte Subpopulations in the Intestinal Lymph Nodes in Goats

The incidence and location of CD2⁺, CD4⁺, CD8⁺ and γ/δ T lymphocytes and IgM⁺ B lymphocytes were studied in the intestinal lymph nodes in 1-week, 1-month, 3-month and 7-month-old goats, using monoclonal antibodies and immuno-histochemical methods. The cortical area of the intestinal lymph nodes in 1-week-old animals contains only primary follicles occupied by IgM⁺ B lymphocytes and some CD2⁺CD4⁺ T lymphocytes. In goats older than 1 month, secondary follicles, that increased in number and size with age, were observed; the light zone of the germinal centre was occupied by IgM⁺ lymphocytes and some CD2⁺ and CD4⁺ T lymphocytes. In the other compartments of the lymph nodes, B lymphocytes were scarce, their number increasing with age in the medulla and diminishing in the paracortex. The numerous CD2⁺ T lymphocytes in the interfollicular area increased in number in the paracortical area of the 7-month-old goats, simultaneously with an increase in the MHC II⁺ dendritic cells and the CD4/CD8 ratio, which was greater than 1. The γ/δ T lymphocytes represented a minor subpopulation scattered through the lymph nodes.     

Engraftment of canine peripheral blood lymphocytes into nonobese diabetic-severe combined immune deficient IL-2R common gamma chain null mice

To study the canine immune system we generated a mouse model engrafted with canine lymphocytes using NOD SCID IL2R common gamma chain ?/? (NSG) mice as recipients (Ca-PBL-SCID). Engraftment of canine peripheral blood lymphocytes (PBLs) was determined post-injection with 10⁷ peripheral blood mononuclear cells (PBMCs) into irradiated NSG mice using flow cytometry and fluorescently labeled antibodies specific to canine helper T cells (CD45⁺ CD4⁺), cytotoxic lymphocytes (CD45⁺ CD8⁺), regulatory T cells (CD45⁺ CD4⁺ Foxp3⁺), and B cells (CD45⁺ Ig⁺ CD21_lo). Canine CD45⁺ lymphocytes were detectable as early as day 1 in the peritoneal cavity, and beginning at 9 days in the blood, bone marrow, and spleen. CD4⁺ T cells, of which Foxp-3⁺ CD25_hi cells constituted a minor percentage, were the predominant lymphocyte population at 9 days post engraftment contrasting with increasing proportions of CD8⁺ CTL's and Ig⁺ B cells beginning at 16 days. Canine immunoglobulin was initially detected in the serum of Ca-PBL-SCID mice at 9 days post-engraftment and peaked in concentration at day 36. From day 28 to 52 post-engraftment 30% of the Ca-PBL-SCID mice became markedly anemic and thrombocytopenic, yet gross and histopathologic examination of bone marrow, kidneys, spleen, liver, and intestine revealed no obvious lesions. Blood smear evaluation revealed agglutination of mature red blood cells, reticulocytes and a regenerative anemia. These findings demonstrate that NSG mice are capable of engraftment of canine PBLs yet develop graft versus host disease similar to Hu-PBL-SCID mice.