Definition of the specificity of monoclonal antibodies against porcine CD45 and CD45R: report from the CD45/CD45R and CD44 subgroup of the Second International Swine CD Workshop

Swine cell binding analyses of a set of 48 monoclonal antibodies (mAbs), including eleven standards, assigned to the CD44 and CD45 subset group of the Second International Swine CD Workshop yielded 13 clusters. Although none of these corresponded to CD44, seven mAbs formed a cluster which was identified as being specific for restricted epitopes of CD45 (CD45R). In addition, a T-cell subset specific cluster comprised of four mAbs was also identified. Two mAbs (STH106 and SwNL 554.1) reacted exclusively with CD8 bright lymphocytes, the other two (2B11 and F01G9) with a subset of CD4 lymphocytes. The other 10 clusters were either specific for MHC-class I like molecules or overlapped with clusters identified by the adhesion molecule subgroup and are therefore just briefly discussed in this report. The specificity of all the mAbs in the CD45R cluster was verified by their ability to immunoprecipitate distinct proteins and to react with CHO cells expressing individual isoforms of CD45. Three CD45R mAbs (3a56, MIL5, −a2) did react with a 210 kDa isoform(s), while another three (STH267, FG2F9, 6E3/7) only recognized a 226 kDa isoform(s). The remaining one (MAC326) precipitated both a 210 and 226 kDa protein. The specificity of all the mAbs in the CD45R cluster, and of the CD45 common mAbs, was confirmed by their reactivity with CHO cells transfected with cDNAs encoding the extracellular and transmembrane portions of distinct CD45R isoforms. Those mAbs recognizing a 210 kDa protein reacted with CHO cells expressing the CD45RC isoform, while those capable of precipitating a 226 kDa, but not the 210 kDa, polypeptide recognized CHO cells expressing either the CD45RAC and the relatively rare CD45RA isoform. MAC326 was unique in its inability to react with CHO cells engineered to produce the CD45RC and CD45RAC isoforms. Thus, three mAbs (6E3/7, STH267, and FG2F9) appear to be specific for an epitope(s) encoded by the A exon, while one (MAC326) recognizes a determinant encoded by the C exon. The remaining three mAbs (3a56, −a2, MIL5) are apparently specific for an epitope(s) which results from the fusion of the C exon to the invariant leader sequence and is destroyed by inclusion of the A exon. All three CD45 common mAbs, K252.1E4, MAC323 and 74.9.3, did react with the CHO cells lines expressing either the CD45RA, CD45RC, CD45RAC or CD45RO isoforms, but not with untransfected CHO cells. When the natural expression of CD45 isoforms was examined by reacting lymphocytes with CD45R mAbs, a high level expression of isoforms containing the A exon-generated domain was detected in all B cells while the majority of CD4⁺ T cells had undetectable or lower expression density of this protein than B cells. In contrast, the density of expression of the CD45 isoform(s) containing the C exon-generated domain ranged from undetectable to high in CD4⁺ T cells whereas the amounts were approximately ten-fold lower in B cells. Overall this panel of CD45 mAbs will be very useful in analyzing the maturation and differentiation of swine lymphoid cells subsets.     

Polymorphism of the CD4 and CD5 differentiation antigens in cattle.

Monoclonal antibodies (mAbs) specific for bovine CD4 and CD5 antigens have been found to identify polymorphic determinants on these molecules. In the case of CD5, mAb IL-A67 recognises one allotypic form of the antigen while four other CD5-specific mAbs in the workshop (CC17, CC29, BLT-1 and 8C11) recognise a second allotype. The CD4-specific mAbs submitted to the workshop reacted with the cells of all animals tested. However, a further two mAbs (CC26 and IL-A18) specific for CD4 were found to react with cells only from about 85% of animals tested. Sequential immuno-precipitation experiments together with family studies showed that the allotypes of CD4 and CD5 are both inherited in a simple Mendelian manner and are co-dominantly expressed. One of the CD5 allotypes was not detected in Bos taurus animals while the gene frequency of the second allotype was only about 10% in the B. indicus animals tested. The gene frequency of the CD4 allotype detected by CC26 and IL-A18 was similar in the two sub-species.     

In vitro effects of dexamethasone on bovine CD25+CD4+ and CD25−CD4+ cells

This paper investigates the in vitro effect of dexamethasone on bovine CD25^highCD4⁺, CD25^lowCD4⁺ and CD25⁻CD4⁺ T cells. Only a small percentage of bovine CD25^highCD4⁺ (2–4%) and CD25^lowCD4⁺ (1–2%) cells expressed Foxp3. Dexamethasone caused considerable loss of CD25⁻CD4⁺ cells, but it increased the relative and absolute numbers of CD25^highCD4⁺ and CD25^lowCD4⁺ lymphocytes, while at the same time reducing the percentage of Foxp3⁺ cells within the latter subpopulations. Considering all these, as well as the intrinsically poor Foxp3 expression in bovine CD25⁺CD4⁺, it can be concluded that the drug most probably increased the number of activated non-regulatory CD4⁺ lymphocytes. It has been found that changes in cell number were at least partly caused by proapoptotic effect of the drug on CD25⁻CD4⁺ cells and antiapoptotic effect on CD25^highCD4⁺ and CD25^lowCD4⁺ cells. The results obtained from this study indicate that the involvement of CD4⁺ lymphocytes in producing the anti-inflammatory and immunosuppressive effect of dexamethasone in cattle results from the fact that the drug had a depressive effect on the production of IFN-γ by CD25⁻CD4⁺ cells. Secretion of TGF-β and IL-10 by CD4⁺ lymphocytes was not involved in producing these pharmacological effects, because the drug did not affect production of TGF-β and, paradoxically, it reduced the percentage of IL-10⁺CD4⁺ cells.     

鸡CD4和CD8分子研究进展

鸡CD4和CD8分子是T细胞表面重要的表面标志,绝大部分胸腺细胞表面都表达CD4和CD8分子,但大多数脾脏和外周血的T细胞表面只表达CD4或CD8分子,或两者都不表达。少数脾脏和外周血中存在的CD4 CD8 T 细胞具有重要的生物学功能。不同品种鸡的CD4 基因具有高度的保守性,而CD8αcDNA 在胞外区表现为多型性。鸡的CD4和CD8分子在组织分布、结构和功能等方面有着很大的相似性。针对鸡CD4 和CD8分子的单克隆抗体为研究这些免疫细胞的生理功能及细胞表面标志的生物学作用等创造了有利条件。     

Influence of sex steroids on the viability and CD11b, CD18 and CD47 expression of blood neutrophils from dairy cows in the last month of gestation

In the period around parturition, cows experience an increased susceptibility for the development of Escherichia coli mastitis. This increased susceptibility has been correlated with a decreased functionality of neutrophils. In the current study, it is suggested that the decreased neutrophil functionality may be induced by the extensive alterations in sex steroid levels occurring around parturition. It was first hypothesized that 17beta-estradiol and progesterone influence the viability, apoptosis and necrosis of blood neutrophils from cows in their last month of gestation. Subsequently, it was hypothesized that 17beta-estradiol modulates the expression of CD11b, CD18 or CD47 thereby explaining its influence on the migration of bovine neutrophils. Neither 17beta-estradiol nor progesterone significantly influenced viability, apoptosis or necrosis in spontaneous apoptosis conditions. However, when apoptosis was induced with TNF-alpha and gliotoxin, progesterone exerted a survival effect (P<0.05). In addition, 17beta-estradiol treatment of bovine blood neutrophils significantly decreased the expression of CD47 (P<0.05) but not of CD11b or CD18. It can be concluded that 17beta-estradiol and progesterone do not affect spontaneous apoptosis of bovine blood neutrophils while a survival effect was observed for progesterone on induced neutrophils apoptosis. Moreover, our results concerning the influence of 17beta-estradiol on the CD11b, CD18 and CD47 expression extend previous demonstrations of the suppressive effect of 17beta-estradiol on neutrophils migration and indicate that the altered expression of CD47 may contribute to this phenomenon.     

In vitro effects of dexamethasone on bovine CD25(+)CD4(+) and CD25(-)CD4(+) cells

This paper investigates the in vitro effect of dexamethasone on bovine CD25(high)CD4(+), CD25(low)CD4(+) and CD25(-)CD4(+) T cells. Only a small percentage of bovine CD25(high)CD4(+) (2-4%) and CD25(low)CD4(+) (1-2%) cells expressed Foxp3. Dexamethasone caused considerable loss of CD25(-)CD4(+) cells, but it increased the relative and absolute numbers of CD25(high)CD4(+) and CD25(low)CD4(+) lymphocytes, while at the same time reducing the percentage of Foxp3(+) cells within the latter subpopulations. Considering all these, as well as the intrinsically poor Foxp3 expression in bovine CD25(+)CD4(+), it can be concluded that the drug most probably increased the number of activated non-regulatory CD4(+) lymphocytes. It has been found that changes in cell number were at least partly caused by proapoptotic effect of the drug on CD25(-)CD4(+) cells and antiapoptotic effect on CD25(high)CD4(+) and CD25(low)CD4(+) cells. The results obtained from this study indicate that the involvement of CD4(+) lymphocytes in producing the anti-inflammatory and immunosuppressive effect of dexamethasone in cattle results from the fact that the drug had a depressive effect on the production of IFN-γ by CD25(-)CD4(+) cells. Secretion of TGF-β and IL-10 by CD4(+) lymphocytes was not involved in producing these pharmacological effects, because the drug did not affect production of TGF-β and, paradoxically, it reduced the percentage of IL-10(+)CD4(+) cells.     

Molecular cloning and characterization of the alphaX subunit from CD11c/CD18 horse integrin

This work reports the cloning and sequence determination of the horse alpha subunit of the integrin CD11c/CD18, a marker of dendritic cells. A cDNA clone of 4582 base pairs was obtained. It encodes a protein segment of 1086 amino acid residues of the extracellular domain with 10 potential sites of glycosylation, a transmembrane domain of 32 residues and a C-terminal cytoplasmic tail of 24 residues. A phylogenetic analysis of this integrin shows close similarity (83%) with that of Canis familiaris.     

Immunohistochemical detection of SWC3, CD2, CD3, CD4 and CD8 antigens in paraformaldehyde fixed and paraffin embedded porcine lymphoid tissue

Identification of the different cell types of the immune system is important for in situ studies on the pathogenesis of infectious diseases in various animals, including the pig. Unfortunately, many monoclonal anti-leukocyte antibodies are only useful for staining frozen tissue sections with inherent poor tissue morphology, and are not readily adapted to formaldehyde fixed and paraffin embedded tissue with well preserved morphology. Seven well characterised monoclonal antibodies against porcine leukocyte antigens were tested on neutral buffered paraformaldehyde fixed and paraffin embedded porcine tissue sections using the highly sensitive tyramide signal amplification system. Combining this method with different antigen retrieval techniques enabled us to detect CD2, CD3, CD4, CD8 and SWC3 antigen expressing cells in porcine lymphoid tissue. Thus, we describe herein methods for the detection of several major cell types of the porcine immune system in fixed tissue with optimal preservation of histological details.     

荧光定量RT-PCR检测鸡CD4、CD8基因表达水平

白细胞分化抗原CD4和CD8在机体免疫应答及其信号传递过程中发挥着重要作用。本试验建立了定量检测鸡CD4和CD8 mRNA表达水平的SYBR Green I实时荧光定量RT-PCR（RRT-PCR）方法,并采用该方法对26-50日龄商品鸡外周血淋巴细胞（PBL）中CD4和CD8 mRNA表达水平进行了检测。结果显示：所建立的RRT-PCR对CD4和CD8 mRNA的扩增效率分别为93%和91%;线性范围分别在10^-4-10^-9和10^-3-10^-9;相关系数分别为0.998 0和0.999 9;最低分别能检测105和120拷贝;熔解曲线分别在78.2和86.7℃附近出现1个单特异峰;组内变异系数分别在1.13%-2.15%和1.17%-3.68%,组间变异系数分别在1.16%-3.25%和1.66%-2.86%。26-50日龄鸡PBL CD4和CD8的mRNA表达水平有小幅波动,与采用流式细胞术检测结果的报道一致。本试验建立的RRT-PCR方法敏感性高、稳定性和再现性好,为检测鸡CD4、CD8基因表达水平提供了精确定量的新方法。     

Increased apoptosis of CD4 and CD8 T lymphocytes in the airways of horses with recurrent airway obstruction

Recurrent airway obstruction (RAO, also known as equine heaves) is an inflammatory condition similar to human asthma caused by exposure of susceptible horses to poorly ventilated stable environments. The disease is characterized by neutrophilic airway inflammation, mucus hypersecretion and reversible bronchoconstriction. This inflammatory process is mediated by several factors, including antibodies, cytokines, resident cells of the airway and inflammatory cellular components that arrive in the respiratory tract. An increasing body of evidence has lent support to the concept that a dysregulation of T cell apoptosis may play a central role in the development of airway inflammation and the associated asthma. Therefore, the aim of this study was to investigate early and late apoptosis of CD4 and CD8 T cell subpopulations obtained from the airways of acute RAO-positive animals after exposure to hay/straw. The percentages of CD4 and CD8 T cells and their associated frequencies of apoptosis were quantified using flow cytometry. Hay/straw exposure induced clinical airway obstruction, airway neutrophilia and increased airway mucus production in RAO-positive horses. In addition, allergen exposure increased the percentage of CD4 T cells in RAO-positive horses as well as the frequency of early and late apoptosis in both CD4 and CD8 lymphocyte subpopulations. These results suggest that the higher frequency of lymphocyte apoptosis may play a role in disease progression of horses afflicted with RAO and may partially explain the characteristic remission of this pathological condition once the allergen source is removed. However, further studies are needed to clarify the role of T cell apoptosis in RAO-affected horses.     

Changes in the population of lymphocytes expressing CD4 and CD8 during the process of atresia of white follicles in hens

The aim of this study was to determine whether the population of lymphocytes expressing CD4 and CD8 molecules changed in the white follicles during atresia in chickens. Frozen sections of healthy, early atretic, advanced atretic and late atretic follicles were immunostained for CD4 and CD8, and the populations of positive cells were analyzed under a light microscope. In the healthy, early atretic and advanced atretic follicles both CD4+ and CD8+ cells were localized in the theca layer, but not in the granulosa layer. However, an influx of CD4+ and CD8+ cells was observed not only in the theca but also in the follicular cavity that was formed by disintegration of the oocyte in late atretic follicles. The frequency of CD4+ T cells in the theca layer did not differ among healthy, early atretic and advanced atretic follicles, but was significantly increased in the late atretic follicles (P < 0.05). The frequency of CD8+ cells showed a pattern of change that resembled that of CD4+ T cells, with a significantly greater population in late atretic follicles than the other follicles (P < 0.05). These results suggest that CD4+ and CD8+ cells are increased in the late atretic follicles, probably to promote the tissue regression.     

AA肉鸡血液CD3+、CD4+和CD8+T淋巴细胞的变化规律

采用流式细胞仪检测1、3、5、7、14、21、28、35、42、49日龄AA肉鸡血液中的CD3、CD4、CD8阳性T细胞比例。研究结果表明:1～5日龄T细胞逐渐进入血液参与细胞免疫,7、21日龄注射疫苗起免疫应答作用,28日龄后基本形成稳固的细胞免疫水平。     

Influence of post-ovulatory insemination on sperm distribution,pregnancy and the infiltration by cells of the immune system,and the distribution of CD2, CD4, CD8 and MHC class II expressing cells in the sow endometrium

This study investigates the distribution of leucocytes, CD2+, CD4+, CD8+ lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium following post-ovulatory insemination in relation to clinical findings and pregnancy outcome. Crossbred multiparous sows were inseminated once either at 15-20 h after ovulation [experiment 1, slaughtered at 20-25 h (5-6 h after artificial insemination (AI), group 1-A, n = 4), at 70 h after ovulation (group 1-B, n = 4), on day 11 (group 1-C, n = 4, first day of standing oestrus = day 1) or on day 19 (group 1-D, n = 4)] or 30 h after ovulation [experiment 2, slaughtered at 5-6 h after AI (group 2-A, n = 4) or on day 19 (group 2-D, n = 3)]. The uterine horns were flushed to control for the presence of spermatozoa and neutrophils and/or for recovery of oocytes and/or embryos. Mesometrial uterine samples were plastic embedded and stained. Cryofixed uterine samples were analysed by immunohistochemistry using mAbs to lymphocyte subpopulations and MHC class II molecules. Light microscopy was used to examine surface (SE) and glandular epithelia (GE), and connective tissue layers, both subepithelially (SL) and glandular (GL). In experiment 1, group 1-A, only one sow had spermatozoa in the utero-tubal junction (UTJ). Marked/moderated numbers of neutrophils and spermatozoa were observed in the flushings of two sows. In group 1-B, altogether 23 of 48 oocytes were cleaved. Day 11 (1-C), embryos with small diameter were observed. Day 19 (1-D), no embryos were found but small pieces of foetal membrane were observed in one of the sows. In group 1-A, large numbers of neutrophils were found within the SE and SL but with high individual variation. For T lymphocyte subpopulations, in the SE, most CD2+ cells were found in group 1-A. For both SE and GE in all groups, the number of CD8+ cells was significantly larger than that of CD4+ cells. In experiment 2, group 2-A, no sow had spermatozoa in the UTJ or in the uterine flushings. At day 19, no sow was pregnant. In group 2-A, large numbers of neutrophils were found within the SE and SL but with high individual variation. At day 19, high E2 levels showed a hormonal prooestrous stage but the endometrial neutrophil infiltration normally expected at pro-oestrus was absent. In conclusion, post-ovulatory insemination (about 18 h after ovulation) resulted in impaired spermatozoa transport within the uterus and embryonic degeneration. In sows post-ovulatory inseminated at a later stage (30 h after ovulation), no sow was pregnant. In both experiments, disturbed immune cell patterns were observed in some individuals.     

呕吐毒素对断奶仔猪外周血液中CD4~+、CD8~+淋巴细胞及抗氧化水平的影响

为了探讨低剂量呕吐毒素对断奶仔猪外周血液中CD4+和CD8+淋巴细胞及抗氧化水平的影响,将用10头平均体重为(7.70±1.00)Kg的三元杂交断奶仔猪,随机分成两组,对照组(基础饲粮)和脱氧雪腐镰刀菌烯醇(DON)组(基础饲粮+DON),试验开始前以及第8、16、24、36天采取仔猪前腔静脉静脉血液检测CD4+、CD8+淋巴细胞和CAT、GSH-Px、SOD、MDA的活性。结果对照组与DON组的CD4+、CD8+淋巴细胞差异都不显著,但是血清CAT、GSH-Px、SOD、MDA的活性都会发生明显变化。2 mg/Kg低剂量DON污染饲粮对断奶仔猪外周血液的CD4+、CD8+淋巴细胞不产生明显影响,对血清中抗氧化体系平衡会产生一定的损害。     

A rapid multiple immunofluorescence method for the simultaneous detection of CD20 and CD3 in canine and feline cytological samples

CD20 and CD3 are considered reliable markers for B and T cells, respectively. This study aimed to develop a rapid multiple immunofluorescence (RMIF) method for the detection of CD20 and CD3 on a single cytology slide. Air-dried smears were prepared using samples collected from dogs (n=26) and cats (n=6). Immunosignal detection using the newly developed method required 60 min. Clear immunosignals for CD20 and CD3 were detected in 24 of 26 samples in dogs and in all 6 cats. As the RMIF (CD20/CD3) method can detect markers of both B and T cells simultaneously on a single cytology smear, it would be an efficient tool for the immunophenotyping of canine and feline lymphoma samples.     

Characterization of monoclonal antibodies assigned to the CD45 subgroup of the Third International Swine CD Workshop.

As a result of the first-round cluster analysis, a panel of 16 novel monoclonal antibodies (mAbs) was assigned for detailed analysis to the CD45 subgroup of the Third International Swine CD Workshop. The specificity of the mAbs was initially determined by examining their reactivity with Chinese hamster ovary (CHO) cells engineered to express individual isoforms of porcine CD45. These analyses indicated that seven of the mAbs (PG77A, PG96A, PG167A, PGB78A, 3C/9, MIL13, NHT 101) recognized the portion of the CD45 molecule encoded by the A exon (CD45RA), while one (MIL15) was specific for that portion encoded by the C exon (CD45RC). In each case, the designation was supported by the demonstration that the molecular weight(s) of the recognized antigen(s) in porcine mononuclear cells, as determined by immunoprecipitation, corresponded to the predicted size(s) according to their specificity. As expected, a similar correlation was obtained for five standard mAbs whose specificity for either common or restricted epitopes of porcine CD45 had been established in previous workshops. Screening of the remaining 174 mAbs that comprised this workshop but were excluded from the CD45 subgroup by cluster analysis failed to detect any additional ones reactive with the porcine CD45-expressing cells.