Generation and characterization of monoclonal antibodies to equine NKp46

The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize full-length NKp46 expressed on the surface of transfected CHO cells as a GFP fusion protein. The mAbs recognized a population of lymphocytes by flow cytometric analysis that was morphologically similar to NKp46+ cells in humans and cattle. In a study using nine horses, representative mAb 4F2 labeled 0.8-2.1% PBL with a mean fluorescence intensity consistent with gene expression data. MAb 4F2+ PBL were enriched by magnetic cell sorting and were found to express higher levels of NKP46 mRNA than 4F2- cells by quantitative RT-PCR. CD3-depleted PBL from five horses contained a higher percentage of 4F2+ cells than unsorted PBL. Using ELISA, we determined that the nine mAbs recognize three different epitopes. These mAbs will be useful tools in better understanding the largely uncharacterized equine NK cell population.     

Cross-species reactivity of seven monoclonal antibodies with equine lymphocytes by flow cytometry

The recognition of equine lymphocyte antigens by monoclonal antibodies (mAbs) directed against human CD11a, CD18, CD21, CD23, CD29 and DR, as well as mouse CD23 was studied by flow cytometry. Unlike anti-CD11a, -CD21, -CD23 and DR mAbs, anti-CD18 and CD29 mAbs labelled the same percentage of horse peripheral blood lymphocytes (PBL) as human PBL. Double-staining with anti-horse immunoglobulin antibodies showed that anti-CD21 and -CD23 mAbs are mainly bound to peripheral blood B lymphocytes. The seven mAbs were also tested on the lymph node and thymus cells. The molecular targets of anti-CD11a, CD18 and CD29 mAbs were confirmed by immunoprecipitation of the membrane proteins. Our results suggest that anti-CD18, -CD29 and -DR mAbs recognise similarly expressed molecular homologues on equine cells, but that anti-CD11a, -CD21 and -CD23 mAbs recognise either different molecules or homologues that are expressed at different levels on horse cells.     

Report on the analyses of mAb reactive with porcine CD8 for the second international swine CD workshop

Based on an analysis of their reactivity with porcine peripheral blood lymphocytes (PBL), only three of the 57 mAbs assigned to the T cell/activation marker group were grouped into cluster T9 along with the two wCD8 workshop standard mAbs 76-2-11 (CD8a) and 11/295/33 (CD8b). Their placement was verified through the use of two-color cytofluorometry which established that all three mAbs (STH101, #090; UCP1H12-2, #139; and PG164A, #051) bind exclusively to CD8⁺ cells. Moreover, like the CD8 standard mAbs, these three mAbs reacted with two proteins with a MW of 33 and 35 kDa from lymphocyte lysates and were, thus, given the wCD8 designation. Because the mAb STH101 inhibited the binding of mAb 76-2-11 but not of 11/295/33, it was given the wCD8a designation. The reactivity of the other two new mAbs in the T9 cluster with the various subsets of CD8⁺ lymphocytes were distinct from that of the other members in this cluster including the standards. Although the characteristic porcine CD8 staining pattern consisting of CD8^low and CD8^high cells was obtained with the mAb UCP1H12-2, a wider gap between the fluorescence intensity of the CD8^low and CD8^high lymphocytes was observed. In contrast, the mAb PG164A, not only exclusively reacted with CD4⁻/CD8^high lymphocytes, but it also failed to recognize CD4/CD8 double positive lymphocytes. It was concluded that this mAb is specific for a previously unrecognized CD8 epitope, and was, thus, given the wCD8c designation. A very similar reactivity pattern to that of PG164A was observed for two other mAbs (STH106, #094; and SwNL554.1, #009). Although these two mAbs were not originally positioned in the T cell subgroup because of their reactivity and their ability to inhibit the binding of PG164A, they were given the wCD8c designation. Overall, five new wCD8 mAbs were identified. Although the molecular basis for the differences in PBL recognition by these mAbs is not yet understood, they will be important in defining the role of CD8⁺ lymphocyte subsets in health and disease.     

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.     

Differences in leukocyte differentiation molecule abundances on domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) neutrophils identified by flow cytometry

Although both domestic sheep (DS) and bighorn sheep (BHS) are affected by similar respiratory bacterial pathogens, experimental and field data indicate BHS are more susceptible to pneumonia. Cross-reactive monoclonal antibodies (mAbs) for use in flow cytometry (FC) are valuable reagents for interspecies comparative immune system analyses. This study describes cross-reactive mAbs that recognize leukocyte differentiation molecules (LDMs) and major histocompatibility complex antigens on DS and BHS leukocytes. Characterization of multichannel eosinophil autofluorescence in this study permitted cell-type specific gating of granulocytes for evaluating LDMs, specifically on neutrophils, by single-label FC. Evaluation of relative abundances of LDMs by flow cytometry revealed greater CD11a, CD11b, CD18 (β₂ integrins) and CD 172a (SIRPα) on DS neutrophils and greater CD14 (lipopolysaccharide receptor) on BHS neutrophils. Greater CD25 (IL-2) was identified on BHS lymphocytes following Concavalin A stimulation. While DS and BHS have similar total peripheral blood leukocyte counts, BHS have proportionately more neutrophils.     

Individual antigens of cattle. Differentiation antigens expressed predominantly on CD4- CD8- T lymphocytes (WC1, WC2).

The 14 mAbs representing workshop cluster 1 recognise a 215/300 kDa antigen expressed on a subpopulation of lymphocytes which express low levels of CD5 but are negative for other B and T cell markers defined by workshop antibodies. Separate studies with cDNA probes for bovine CD3 and T cell receptor indicate that these lymphocytes are gamma/delta T cells. It is of note that the different mAbs react with varying proportions of this cell population, suggesting that the antigen undergoes considerable post-translational modification. A further two mAbs, designated workshop cluster 2, react with a 37/47 kDa heterodimeric molecule expressed in a subpopulation of the WC1+ cells and on an additional small population of T lymphocytes. The cell populations recognised by the two mAbs are different although they overlap in some animals. It is suggested that these mAbs may be specific for T cell receptor molecules.     

Reactivity of cross-reacting monoclonal antibodies with canine leukocytes, platelets and erythrocytes

A panel of 380 commercially available monoclonal antibodies (mAbs) against human CD molecules from various sources was tested during the 8th Human Leukocyte Differentiation Antigen Workshop (HLDA8) for cross-reactivity on canine peripheral blood leukocytes by flow cytometry. In addition, all mAbs were used to label a 50:50 mixture of platelets and erythrocytes of the same dogs. This testing resulted in 51 cross-reacting mAbs. mAbs with specificity for CD9, CD29, CD42a, CD61, and CD41/CD61 showed cross-reactivity with canine platelets in a non-polymorphic and one mAb with the erythrocyte antigen CD235a in a polymorphic reaction pattern. Canine leukocyte-reactive mAbs included those with specificity for CD11a, CD11b, CD14, CD18, CD21, CD22, CD47, CD49d, CD49e, CD56, CD62L, CD91, CD94, and CD172a. In addition, several mAbs resulted in a staining pattern of canine cells which suggest that the canine epitope equivalents have an alternate expression pattern from that expected for humans (CD1a, CD35, CD44, CD45, CD75s, CD81). In summary, this study confirmed the reactivity of previously described cross-reactive mAbs with canine cells and resulted in the characterization of mAbs recognizing so far undetectable canine CD molecules.     

Cross-reactivity of mAbs to human CD antigens with cells from cattle

A panel of 377 commercially available mAbs were submitted to the animal homologue section of the 8th International Workshop on Human Leukocyte Differentiation Antigens (HLDA8, Adelaide, Australia) for cross-reactivity studies on different animal species. In this study we describe the results of testing the mAbs on cattle cells by flow cytometry and Western blot. Eight commercial suppliers participated, providing mAbs to a total of 144 CD antigens plus controls. Fifty-two mAbs were identified as potentially staining cattle cells in the first round screen. In the second phase, 38 mAbs were confirmed as staining cattle cells. This included some that may recognise polymorphic determinants and others with atypical distribution patterns compared to humans. mAb to human CD9, CD11a, CD14, CD18, CD21, CD23, CD29, CD44, CD45R, CD47, CD49d and CD172a cross-reacted with bovine cells and mAb to CD22, CD88, CD119 and CD163 stained CD antigens that have not previously been identified in cattle.     

Development and characterization of mouse monoclonal antibodies reactive with chicken CD83

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD83 (chCD83), a membrane-bound glycoprotein belonging to the immunoglobulin superfamily that is primarily expressed on mature dendritic cells (DCs). A recombinant chCD83/IgG4 fusion protein containing the extracellular region of chCD83 was expressed in Chinese Hamster Ovary (CHO) cells and isolated from the spent cell culture medium by protein G affinity chromatography. The extracellular region of the chCD83 protein was purified and used to immunize mice. A cell fusion was performed, from which 342 hybridomas were screened for mAbs to chCD83. Two mAbs, chCD83-159 and chCD83-227, stained the greatest percentage of chCD83-transfected CHO cells and were selected for further characterization. By flow cytometry, both mAbs reacted with a chicken macrophage cell line, HD11. Both mAbs also recognized a single 53 kDa protein on Western blots of lysates from lipopolysaccharide-stimulated spleen mononuclear cells or unstimulated HD11 cells. Immunostaining of chicken secondary lymphoid organs identified chCD83(+) cells with morphologic and subtissue localization properties comparable to mammalian DCs. In vitro stimulation of spleen mononuclear cells with concanavalin A (Con A) decreased the percentage of chCD83(+) cells compared with cells treated with medium alone. Interestingly, spleen cells treated with Con A in the presence of chCD83-227 mAb exhibited decreased percentage of MHCII(+) cells compared with cells treated with an isotype-matched negative control mAb. These chCD83 mAbs may be useful for future investigations of chicken immune cell maturation and mechanisms of action.     

Use of flow cytometry to identify monoclonal antibodies that recognize conserved epitopes on orthologous leukocyte differentiation antigens in goats, llamas, and rabbits

Flow cytometry was used to screen a panel of 320 mAbs, submitted to the Animal Homologues Section of the HLDA8, for mAbs that recognize epitopes conserved on orthologous leukocyte differentiation antigens (LDA) in goats, lamas, and rabbits. Nineteen mAbs specific for CD11a (1), CD14 (3), CD18 (1), CD21 (1), CD29 (2), CD44 (2), CD47 (3), CD49d (1), CD172a (1), CD45RB (1), CD61 (1), RACT48A, and GBSP71A reacted with goat LDA. Twenty three mAbs specific for CD7 (1), CD9 (2), CD11a (1), CD14 (3), CD18 (4), CD29 (1), CD32 (1), CD44 (1), CD47 (4), CD49d (2), CD50 (1), CD80 (1), CD172a (1), and GBSP71A reacted with llama LDA. Eighteen mAbs specific for CD9 (2), CD11a (1), CD14 (2), CD18 (4), CD21 (1), CD44 (2), CD45RB (1), CD49d (1), CD209 (1), RACT48A, and GBSP71A reacted with rabbit LDA. The specificities of two cross reactive mAbs that recognize different conserved epitopes on all leukocytes in two species (RACT48A) and all three species (GBSP71A) have not been determined. The patterns of reactivity of most of the mAbs were consistent with patterns of reactivity noted on human leukocytes. The specificity of some cross reactive mAbs generated in non-human species were validated on human leukocytes. Further studies are needed to verify that CD7, CD32, CD45RB, CD50, and CD209 recognize orthologous molecules in the indicated species.     

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.     

Use of flow cytometry to develop and characterize a set of monoclonal antibodies specific for rabbit leukocyte differentiation molecules

Flow cytometry was used to identify and characterize monoclonal antibodies (mAbs) that react with rabbit leukocyte differentiation molecules (LDM). Screening sets of mAbs, developed against LDM in other species, for reactivity with rabbit LDM yielded 11 mAbs that recognize conserved epitopes on rabbit LDM orthologues and multiple mAbs that recognize epitopes expressed on the major histocompatibility class I or class II molecules. Screening of mAbs submitted to the Animal Homologues Section of the Eighth Human Leukocyte Differentiation Workshop yielded 7 additional mAbs. Screening of mAbs generated from mice immunized with leukocytes from rabbit thymus or spleen or concanavalin A activated peripheral blood and/or spleen lymphocytes has yielded 42 mAbs that recognize species restricted epitopes expressed on one or more lineages of leukocytes. Screening of the anti-rabbit mAbs against leukocytes from other species yielded one additional mAb. The studies show that screening of existing sets of mAbs for reactivity with rabbit LDM will not be productive and that a direct approach will be needed to develop mAbs for research in rabbits. The flow cytometric approach we developed to screen for mAbs of interest offers a way for individual laboratories to identify and characterize mAbs to LDM in rabbits and other species. A web-based program we developed provides a source of information that will facilitate analysis. It contains a searchable data base on known CD molecules and a data base on mAbs, known to react with LDM in one or more species of artiodactyla, equidae, carnivora, and or lagomorpha.     

Analysis of workshop antibodies to null cells on a non-T/non-B lymphocyte population

Non-T and non-B lymphocytes (null cells) were obtained by Sephadex G-10 depletion followed by treatment with mAbs to CD2 and MHC class II and complement. The enriched cells were principally CD5dim and contained greatly increased numbers of null lymphocytes. This methodology will be useful for null lymphocyte enrichment in examining cell surface molecules and functional attributes of null lymphocytes.     

Workshop studies on monoclonal antibodies in the myeloid panel with CD11 specificity.

Several putative anti-human and swine CD11-specific monoclonal antibodies (mAbs) were included in the myeloid section of the Third International Swine CD Workshop. Failure of clustering analysis to group these mAbs together prompted additional analyses to define the specificities of these mAb. Combination of one and two-color flow cytometry (FCM) and immunoprecipitation (IP) allowed the definition of the mAb into three CD11 groups. Cellular distribution of the molecules recognized by anti-human CD11b and c mAbs on swine cells proved to be significantly different from that found in humans.     

Reactivity of monoclonal antibodies to human CD antigens with cells from mink

Three hundred and seventy six monoclonal antibodies (mAbs) raised against human leukocyte surface antigens were analyzed by flow cytometry for cross reactivities against mink leukocytes. We found 53 mAbs (14%) to cross react. This study defined cross reactions to the following human markers: CD1a, CD9 (4 mAbs), CD10, CD11a (2 mAbs), CD14 (3 mAbs), CD18 (5 mAbs), CD20 (atypical reaction), CD21, CD25 (atypical reaction), CD29 (3 mAbs), CD32, CD41, CD42a, CD44 (4 mAbs), CD45, CD45RO, CD47 (2 mAbs), CD49d (3 mAbs), CD61 (2 mAbs), CD62P, CD66abcd, CD71, CD75s, CD79b (2 mAbs), CD86, CD88, CD104 (atypical reaction), CD172a, CD236R (glycophorin C, (atypical reaction)), Xg(a) carbohydrate antigen, Rhesus antigen and two unspecified PAN-reactive mAbs. In order to characterize the molecular mass of the corresponding cross reacting mink markers, the mAbs were used to immunoprecipitate the surface antigens. Fourteen mAbs out of the 53 mAbs reactive with mink leukocytes gave reproducible IP findings. The masses of the precipitated antigens were generally in good agreement with those of the homologous human markers. We also performed immunohistochemical staining analyses on formalin fixed, paraffin embedded mink tissue from lymph node and spleen, and found 7 out of 22 mAbs to give a positive signal. Generally, the immunohistological analyses resulted in expected staining patterns.     

Characterization of a canine CD44 specific monoclonal antibody

Monoclonal antibodies (mAbs) were generated against a CD44 mRNA expressing (RT-PCR) macrophage/monocyte cell line (DH82) from a dog with malignant histiocytosis. The mAbs, that reacted with DH82 cells by FACS analysis were tested on formalin-fixed, paraffin-embedded tissues. Exclusively the incubation of DH82 cell pellets with mAbs from clone 2D10 resulted in a cell membrane associated immunoreaction. Immunoelectron microscopy specified, that the antibody bound exclusively to the cell membrane and processes of DH82 cells. The mAb was tested on a variety of normal canine tissues, including lymphoid, urinary, alimentary, respiratory, and endocrine organs, nervous tissues, liver, pancreas, skin, and muscles. Furthermore, tumour and inflamed tissues were tested for immunoreaction with the mAb. Immunohistologically, the 2D10 mAb reacted with macrophages/monocytes, subsets of lymphocytes, epithelial cells, and central nervous system white matter. FACS analysis of canine peripheral blood leukocytes showed, that a high proportion of lymphocytes and granulocytes were positive with this mAb. Western blot analysis revealed, that the 2D10 mAb bound to a protein with a molecular weight of about 85 kDa. The results of FACS and Western blot analyses, RT-PCR, immunohistology and immunoelectron microscopy strongly suggest that the antigen detected by the 2D10 mAb is most likely the canine equivalent of human CD44, a cell bound hyaluronan binding proteoglycan.     

Analyses of monoclonal antibodies reacting with porcine CD5: results from the Second International Swine CD Workshop

Among the 57 monoclonal antibodies analyzed within the T-cell group, three mAbs fell within cluster T13 including the CD5a standard b53b7 (No. 174). The two new mAbs 1H6/8 (No. 058) and BB6-9G12 (No. 166) both precipitated 55 and 60 kDa proteins that were of similar molecular weights as the standard. Staining patterns on the various cell types were similar. Both new antibodies inhibited the binding of the CD5a reference mAbs b53b7 to peripheral lymphocytes. These mAbs, therefore both react with the CD5a epitope bringing the number of anti-porcine CD5 mAbs to eight, all of which appear to recognize the same epitope.     

Production and characterization of monoclonal antibodies against porcine interleukin-4

Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. In the present study, recombinant thioredoxin (Trx)-porcine IL-4 (pIL-4) fusion protein was prepared by Escherichia coli (E. coli), and by using this protein as an immunogen, monoclonal antibodies (mAbs) against pIL-4 were produced to establish a basis for a research on immune responses in pigs. Six stable hybridoma cell lines were successfully established and specific binding of each mAb to recombinant pIL-4 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that the subclass of 5 out of 6 mAbs was IgG1 and the rest was IgG2b. Further, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to 4 different epitopes. The recombinant proteins and mAbs produced in this study will be useful tools for the assessment of porcine immune system.     

Screening of anti-human leukocyte monoclonal antibodies for reactivity with equine leukocytes

Three hundred and seventy-nine monoclonal antibodies (mAbs) against various human CD molecules supplied to the HLDA8 animal homologues section (including four isotype controls) were analysed for cross-reactivity with equine leukocytes. First, flow cytometric identification of positively reacting mAbs was performed in one laboratory. Thereafter, a second round of flow cytometric evaluation was performed, involving three laboratories participating in the study. The first test-round indicated 17 mAbs as potentially positive. After the second round of flow cytometric analysis, 14 mAbs remained (directed against CD2, CD11a, CD18, CD44, CD45, CD49d, CD91, CD163 and CD172) where cross-reactivity was anticipated based on similarities between the human and equine staining pattern. Additionally, there was 1 mAb with weak likely positive reactivity, 12 mAbs with positive staining, which likely do not reflect valuable data, 5 mAbs with clear alternate expression pattern from that expected from humans, 5 mAbs with a questionable staining pattern itself, i.e. that was variable between the three labs, 32 mAbs with weak-positive expression and alternate staining pattern, and 279 negative mAbs (including the four isotype controls) were detected. In 31 cases, more appropriate target cells, such as thymocytes or stem cells, were not available for the screening. The results underline the value of this "cross-reactivity" approach for equine immunology. However, as only a few mAbs against leukocyte surface antigens reacted positively (approximately 4% of the mAbs submitted), the analysis of further anti-human mAbs and directed efforts to develop species-specific anti-CD mAb are still required.