Feline large granular lymphocyte (LGL) lymphoma with secondary leukemia: primary intestinal origin with predominance of a CD3/CD8(alpha)(alpha) phenotype

Clinicopathologic and immunophenotypic characteristics of large granular lymphocyte (LGL) neoplasia in 21 cats were examined. All cats were domestic short (19) or long hair (2) with a mean age of 9.3 years at diagnosis. Increased peripheral blood LGL counts were present in 18/21 cats. Neutrophilia (12/21 cats) and increased serum liver enzymes (7/12), total and direct bilirubin (7/13), BUN (5/14), and creatinine (2/14) were observed. Cats usually presented with advanced disease and none survived longer than 84 days (mean 18.8 days) postdiagnosis. Cytologically, LGLs had a mature (6/21), immature (13/21), or mixed (2/21) morphology. Necropsy lesions consisted of neoplastic lymphoid infiltrates in the jejunum, ileum, and duodenum in decreasing order of frequency. In the small intestine, mucosal ulceration (9/13) and epitheliotropism of neoplastic cells (9/13) were common. Neoplastic infiltrates were also present in the mesenteric lymph nodes (13/13), liver (12/13), spleen (8/13), kidneys (5/7), and bone marrow (5/7). A T cell phenotype (CD3epsilon+) characterized LGL neoplasia in 19/21 cases. A CD8alphaalpha+ cytotoxic/suppressor phenotype was present in 12/19 T cell tumors, 2 had a CD4+CD8alphaalpha phenotype, 3 had a CD4-CD8- phenotype, and 2 were CD4+ helper T cells. CD8beta chain expression was not detected in any instance. In two cats, a B or T cell origin could not be established. CD103 was expressed by 11 of 19 (58%) of the lymphomas tested. The immunophenotypic features shared by neoplastic LGLs in the cat and feline intestinal intraepithelial lymphocytes (IELs) support a small intestinal IEL origin for feline LGL lymphoma.     

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.     

CD56 is expressed exclusively on CD3+ T lymphocytes in canine peripheral blood

CD56+ cells in canine blood leukocytes were characterized by flow-cytometric analysis of peripheral blood of 30 healthy adult beagle-dogs (15 males and 15 non-pregnant females). In 19 of the 30 dogs, anti human CD56 antibody, Leu-19, reacted with 8.8-21.7% of peripheral blood lymphocytes. All CD56+ cells simultaneously expressed CD3 molecules on their surface. Further phenotypic analysis revealed that 50.6+/-13.1% of the CD56+ cells showed CD4-CD8+ phenotype and 43.7+/-10.1% showed CD4+CD8- phenotype. Expression intensity of CD56 on the CD4-CD8+CD56+ cells was significantly higher than that on CD4+CD8-CD56+ cells (P<0.001). These findings indicate that CD56, which is a neural cell adhesion molecule, is uniquely expressed on subsets of T lymphocytes in canine peripheral blood.     

Lymphocyte development in fetal piglets: facts and surprises

The developing porcine fetus offers an excellent opportunity for the study of lymphocyte development. Studies on B cell, alphabeta T cells and gammadelta T cells in the last decade have expanded our knowledge of lymphocyte development in pigs. These studies have revealed several interesting differences between swine, mice and humans. For example, porcine peripheral lymphocytes include CD4+CD8+ alphabeta T cells and an abundance of gammadelta T cells that may even prevail over the alphabeta population. There are numerous CD2- gammadelta T cells in the blood and a large number of CD8alphaalpha-bearing cells that include NK cells, conventional gammadelta and alphabeta T cells. All porcine B lymphocytes are CD25(lo) and sIgM+ B cells may differ in the expression of CD2 antigen. Unlike mice, porcine B cells appear approximately 2 weeks before T cells and progenitors undergo VDJH rearrangement at 20th day of gestation (DG20) in the yolk sac and DG30 in the fetal liver before consummating high level lymphogenesis in the bone marrow after DG45. Early B cells show an unexpectedly high proportion of in-frame rearrangements, undergo switch recombination in thymus on DG60 and use N-region insertion from the time of the earliest VDJ rearrangement. The genomic repertoire of VH, DH and JH genes is small compared to mice and humans and swine appear to depend on junctional diversity for the majority of their repertoire. The limited VH repertoire of swine contrasts sharply with the porcine TCRbeta repertoire, which is extensive, extraordinarily conserved and nearly identical to that in humans. Therefore, swine present an example of two highly related receptor systems that have diverged in the same species.     

健康中国恒河猴各淋巴组织中T淋巴细胞表型及分布

试验旨在明确T淋巴细胞在中国恒河猴各组织中的表型与分布,为疾病模型研究提供基础数据。取外周血、腹股沟淋巴结、肠系膜淋巴结及肠道组织,从中分离出淋巴细胞。使用流式细胞术检测分析各种表型的淋巴细胞在组织间的分布。结果表明,淋巴结中CD4⁺ /CD8⁺ T细胞比值高于外周血,肠道固有层中最低,三者差异显著。记忆性T细胞在外周血和肠道固有层T细胞中比重较大,而淋巴结中主要为幼稚T细胞。CD4⁺ T细胞中中心记忆T细胞Tcm为主要亚群,而CD8⁺ T细胞主要为效应记忆细胞Tem。外周血与肠道固有层中增殖T细胞比例相当,而淋巴结中T细胞增殖水平相对较低。各组织中CXCR4受体表达量普遍高于CCR5受体,其中肠道固有层CCR5受体表达水平最高。值得注意的是,有一小群表型CD3⁺ CD4⁺ CD8low的细胞仅在肠道固有层中存在,经分析其功能活性应高于肠道CD4单阳性T细胞。因此,测定了健康中国恒河猴各表型T淋巴细胞在多种淋巴组织中的基础数值,为相关模型研究奠定基础。     

The kinetics of cytokine production and CD25 expression by porcine lymphocyte subpopulations following exposure to classical swine fever virus (CSFV)

Surface expression of IL-2R-alpha (CD25) is widely used to identify activated lymphocyte populations, while interferon-gamma (IFN-gamma) levels have been shown to be a good indicator of cell-mediated immunity (CMI) in pigs. To investigate the relationship between these two parameters, we developed an intracellular cytokine-staining assay and studied the kinetics of cytokine (IFN-gamma and interleukin-10, IL-10) production relative to CD25 expression in porcine lymphocyte subpopulations, following immunization with a classical swine fever (CSF) vaccine. The number of activated memory T cells (CD4(+)CD8(+)CD25(+) cells) increased slightly in the peripheral blood mononuclear cell (PBMC) population soon after vaccination, then diminished within a few weeks. The number of activated cytotoxic T cells (CD4(-)CD8(+)CD25(+) cells) peaked approximately 2 weeks after the memory population. Although the number of IFN-gamma producing cells detected in this experiment was relatively low, the CD4(+)CD8(+) T cells were major IFN-gamma producers in the PBMCs throughout the experiment. In another experiment, CSF-vaccinated pigs were challenged with a virulent classical swine fever virus (CSFV), and the kinetics of CD25 expression and cytokine productions were monitored. Following exposure to the virus, the number of IFN-gamma producing cells in the PBMCs increased markedly in both the vaccinated and unvaccinated groups. The CD4(-)CD8(+) cells were major IFN-gamma producing cells in vaccinated pigs, while both CD4(+)CD8(+) and CD4(-)CD8(+) populations contributed to the IFN-gamma production in the control group. Interestingly, the enhanced IFN-gamma production was not associated with the upregulation of CD25 expression following the CSFV challenge. In addition, exposure to the virulent CSFV significantly increased interleukin-10 production by the CD4(-)CD8(+) populations in PBMCs of the unvaccinated pigs. Taken together, our results indicated that CD25 expression and IFN-gamma production were not tightly associated in porcine lymphocytes. In addition, the CD4(-)CD8(+) lymphocytes of the PBMCs played a major role in cytokine productions following the CSFV challenge.     

Functional characterization of a swine CD4(+)/CD8(+) double positive lymphoblastoid T-cell line with a CD25(+)/CD45RA(-) phenotype generated in vitro with interleukin-2

A dual expressing (CD4(+)/CD8(+)) porcine lymphoblastoid T-cell line (pIL-2d) generated from peripheral blood mononuclear (MN) cells shown to be highly responsive to exogenous interleukin-2 (IL-2) was characterized. The swine MN cells were initially stimulated with concanavalin A (Con A), and sub-passaged using decreasing amounts of conditioned medium (CM), which was prepared from culture fluids of Con A activated porcine MN cells, until a steady growth was observed. The resulting pIL-2d cells require exogenous IL-2 from CM and are highly responsive to recombinant human IL-2 (rhIL-2). The pIL-2d cells exhibited a specific, dose-dependent proliferative response to stimulation with IL-2. The specificity of this proliferative response was confirmed to be IL-2 induced by its inhibition with an anti-swine IL-2 receptor (alpha-swIL-2R) monoclonal antibody (mAb). Furthermore, the pIL-2d cells are highly responsive to exogenous IL-2 contained in culture fluids derived from antigen-driven blastogenic tests performed with lymphocytes of vaccinated swine. This property makes the pIL-2d cells an ideal functional adjunct to immunochemical or molecular tests that are commonly used to measure total porcine IL-2. Interestingly, the phenotype of the pIL-2d cells after five or more passages was shown by flow cytometric analysis to be CD4(+)/CD8(+)/CD45RA(-)/CD25(+) and to remain unchanged thereafter. Although, the mechanism of selection and maintenance of the CD4(+)/CD8(+) DP cells developed here remains unclear, our data suggest that an oligoclonal or polyclonal expansion and maintenance of cells of this phenotype was mediated by exogenous IL-2.     

Cytokine profiles of peripheral blood and airway CD4 and CD8 T lymphocytes in horses with recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is thought to result from an aberrant immune response to inhaled antigens, modulated by T lymphocytes via the secretion of pro-inflammatory cytokines. However data relating to the phenotypes of the T lymphocytes present in peripheral blood and bronchoalveolar lavage fluid of RAO horses and their cytokine profiles are contradictory. The aim of this study was to further investigate the cytokine (IL-4, IL-5, IL-13 and INF-gamma) mRNA expression profile in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes from RAO and control horses, before and at 48 h after horses were exposed to hay/straw. In contrast to previous studies, cytokine expression was quantified in populations of CD4 and CD8 T lymphocytes which were purified using magnetic bead antibody cell separation. Hay/straw exposure induced clinical airway obstruction, airway neutrophilia and airway lymphocytosis in RAO horses, and, induced a mild, but significant, airway neutrophilia in controls. However, hay/straw exposure had no significant effect on peripheral blood lymphocyte or bronchoalveolar lavage lymphocyte cytokine expression in either group. In conclusion, RAO was not associated with alterations in lymphocyte cytokine expression that are consistent with Th1 or Th2 responses, but rather with a general down-regulation in expression of the measured cytokines in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes.     

猪瘟病毒感染对猪外周血T淋巴细胞亚群及TNF-α和IFN-γ的影响

为探讨猪瘟病毒(CSFV)弱毒株T株和野毒株G株感染对猪外周血T淋巴细胞亚群、TNF-α和IFN-γ的影响,本研究应用流式细胞术和ELISA等方法检测CSFV感染猪与未感染猪的白细胞凋亡、CD4+与CD8+T淋巴细胞亚群数量的动态变化以及TNF-α和IFN-γ的动态变化。结果表明,猪感染CSFV T株和G株第4 d和第7 d后CD4+T淋巴细胞比例分别为28.6%、26%和26%、20%,未感染前分别为33.4%和36.8%。猪感染CSFV T株和G株第4 d、第7 d后CD8+T淋巴细胞比例分别为41%、32%和38%、25%,感染前分别为43.8%和48.8%。外周血白细胞凋亡的检测结果显示,猪感染CSFV T株和G株第7 d后,白细胞凋亡比例分别为8.35%和9.89%,未感染的猪为1.63%。ELISA检测结果表明,猪感染CSFV T株和G株第7 d后,TNF-α的产生量分别为553.4 pg/mL和594.2 pg/mL;IFN-γ的产生量分别为8.2 pg/mL和9.8 pg/mL,未感染猪分别为498 pg/mL、12.5 pg/mL。以上结果提示,CSFV感染会引起机体免疫相关细胞及免疫分子发...     

Increased expression of fractalkine and its receptor CX(3)CR1 in canine inflammatory bowel disease and their possible role in recruitment of intraepithelial lymphocytes

The interaction between fractalkine/CX(3)CL1 and its receptor CX(3)CR1 has been reported to play an important role in various human inflammatory diseases, including inflammatory bowel disease (IBD) mediated by lymphocyte chemoattraction. The objective of this study was to investigate the role of fractalkine and CX(3)CR1 in lymphocyte migration in canine IBD. IBD was diagnosed in 34 dogs, and 19 healthy beagles were used as normal controls. We quantified intestinal mRNA and protein expression of fractalkine and CX(3)CR1 by real-time RT-PCR and ELISA, respectively, and examined the localization of fractalkine in canine intestine by immunohistochemistry. The expression of CX(3)CR1 and surface antigens on peripheral blood mononuclear cells (PBMCs) and intraepithelial lymphocytes (IELs) was analyzed by flow cytometry. Intestinal fractalkine and CX(3)CR1 mRNA was significantly up-regulated in IBD dogs compared with the healthy control dogs. In addition, fractalkine expression on intestinal epithelial cells was significantly increased in the intestinal mucosa of IBD dogs compared with the healthy dogs. CX(3)CR1(+) PBMCs were significantly elevated in IBD dogs and positively correlated with the histopathological severity of IELs infiltration. These CX(3)CR1(+) PBMCs predominantly expressed markers for cytotoxic T cells. Almost all IELs expressed CD3, and the majority of cells expressed CD8 rather than CD4, which was analogous to the CX(3)CR1(+) PBMCs. These results suggest that the fractalkine-CX(3)CR1 interaction may contribute to the pathogenesis of canine IBD through migration of IELs.     

Flow cytometric analysis of canine umbilical cord blood lymphocytes

Lymphocyte subsets in canine umbilical cord blood were flow cytometrically analyzed and compared with those of the dams' peripheral blood. The proportion of CD3+ T lymphocytes, CD21+CD3- B lymphocytes, and CD3-CD21- non-T non-B lymphocytes in umbilical cord blood was 52.9%, 30.4%, and 16.7%, respectively. T lymphocyte/B lymphocyte ratio was significantly lower in the umbilical cord blood than in the dams' peripheral blood (2.1 +/- 1.4 versus 11.0 +/- 8.1, P < 0.001). In contrast, CD4+ lymphocyte/CD8+ lymphocyte ratio was significantly higher in the umbilical cord blood than in the dams' peripheral blood (7.6 +/- 2.2 versus 1.8 +/- 0.6, P<0.001). These findings clarified the phenotypic characters of canine umbilical cord blood lymphocytes.     

Longitudinal evaluation of CD4+ and CD8+ peripheral blood and mammary gland lymphocytes in cows experimentally inoculated with Staphylococcus aureus.

Staphylococcus aureus is a major pathogen associated with mastitis, a disease affecting both women and dairy cows. The longitudinal profiles of bovine peripheral blood and mammary gland lymphocyte phenotypes in response to S. aureus-induced mastitis were investigated in dairy cows. Increased percentage of CD4 lymphocytes in the mammary gland between 1 and 8 days post-inoculation, increased milk CD4 protein density per cell between 1-8 days post-inoculation, and a statistically significant negative correlation between post-inoculation bacterial counts in milk and blood lymphocyte CD4 protein density were found. Together with blood and milk leukocyte counts, the milk lymphocyte CD4/CD8 ratio and the milk lymphocyte CD4 protein density were more informative indicators than milk somatic cell counts and bacteriology for identification of early vs. late inflammatory phases. These findings suggest that CD4+ lymphocytes play a protective role in the early stages of S. aureus-induced mastitis.     

Horses with equine recurrent uveitis have an activated CD4+ T‐cell phenotype that can be modulated by mesenchymal stem cells in vitro

Equine recurrent uveitis (ERU) is an immune‐mediated disease causing repeated or persistent inflammatory episodes which can lead to blindness. Currently, there is no cure for horses with this disease. Mesenchymal stem cells (MSCs) are effective at reducing immune cell activation in vitro in many species, making them a potential therapeutic option for ERU. The objectives of this study were to define the lymphocyte phenotype of horses with ERU and to determine how MSCs alter T‐cell phenotype in vitro. Whole blood was taken from 7 horses with ERU and 10 healthy horses and peripheral blood mononuclear cells were isolated. The markers CD21, CD3, CD4, and CD8 were used to identify lymphocyte subsets while CD25, CD62L, Foxp3, IFNγ, and IL10 were used to identify T‐cell phenotype. Adipose‐derived MSCs were expanded, irradiated (to control proliferation), and incubated with CD4⁺ T‐cells from healthy horses, after which lymphocytes were collected and analyzed via flow cytometry. The percentages of T‐cells and B‐cells in horses with ERU were similar to normal horses. However, CD4⁺ T‐cells from horses with ERU expressed higher amounts of IFNγ indicating a pro‐inflammatory Th1 phenotype. When co‐incubated with MSCs, activated CD4⁺ T‐cells reduced expression of CD25, CD62L, Foxp3, and IFNγ. MSCs had a lesser ability to decrease activation when cell‐cell contact or prostaglandin signaling was blocked. MSCs continue to show promise as a treatment for ERU as they decreased the CD4⁺ T‐cell activation phenotype through a combination of cell‐cell contact and prostaglandin signaling.     

Functional characteristics of porcine peripheral T cells stimulated with IL-2 or IL-2 and PMA

In human or mouse, mature T cells express either CD4 or CD8, resulting in different functions in the periphery. Interestingly, porcine CD4 and CD8 double positive (DP) T cells are present in the blood, and their proportions change from youth to adulthood. However, the features of these cells in swine are poorly understood. We investigated the fate of porcine peripheral T cells based on their functional characteristics, including proliferation and the expression of CD4 and CD8 co-receptors. The results showed that all the populations changed their CD8 expression in a time-dependent manner and porcine T cells had different proliferative pattern from human T cells. The results further revealed that Th2 cytokines were increased later in porcine T cells compared to human T cells upon stimulation with IL-2 + PMA. Collectively, we found that the fate of porcine peripheral T cells is different from that of human T cells, and the changes occur in a time- and stimulation-dependent manner.     

Lymphocyte subpopulations in peripheral blood of sheep experimentally infected with tick-borne fever.

The lymphocyte subpopulations in the peripheral blood of normal sheep and sheep experimentally infected with Cytoecetes phagocytophila, the causative agent of tick-borne fever, were analysed by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with tick-borne fever was characterised by a significant reduction in the total number of circulating lymphocytes six days after experimental infection (P less than 0.001). This lymphocytopenia was associated with a significant reduction in the number of B (LCAp220+) and T (CD5+) lymphocytes (P less than 0.001) but there was a significant increase in the number of cells which were neither T nor B (CD5-LCAp220-) cells (P less than 0.01). The reduction in the number of T lymphocytes was due to reduced numbers of circulating CD4+ (helper) T cells, CD8+ (cytotoxic/suppressor) T cells and those with the pan T cell marker (CD5+) but without CD4 or CD8 epitopes (CD4-CD8-). All lymphocytes returned to preinoculation levels 13 to 16 days after experimental infection.     

Changes in peripheral lymphocyte subsets in the type 1 diabetic dogs treated with insulin injections

Plasma metabolites and peripheral lymphocyte subsets were measured in ten diabetic and ten control dogs to investigate their significances as indicators to evaluate immune states in the diabetic dogs. Diabetic dogs were treated with insulin injections, however their plasma glucose and fructosamine concentrations were significantly higher than those of the controls. There were no significant differences in counts of total white blood cells (WBC) and lymphocyte CD8(+) cells (cytotoxic T cells) between the control and the diabetic dogs. In the diabetic dogs, the counts of CD3(+) (T cells), CD4(+) (Helper T cells) and CD21(+) (B cells) cells and the peripheral lymphocytes CD4/CD8 ratio were significantly lower than those in the control dogs. We confirmed abnormality of lymphocyte subsets in insulin treated diabetic dogs and it may relate to depression of immunocompetence and high susceptibility to common infectious diseases.