Leukocyte Differentiation Antigens Research Articles

Monoclonal antibodies to mouse cell-surface antigens.

The cluster of differentiation (CD) nomenclature, originally devised by the International Workshop on Human Leukocyte Differentiation Antigens (HLDA) to classify leukocyte surface antigens, has become more accepted by researchers in nonhuman animal models. The CD homologues from other species are usually identified by multiple studies, including biochemical, functional analysis, cloning, and immunological assays. This unit compiles a complete (as of August, 2001) list of monoclonal antibodies (mAb) to the mouse CD antigens and several non-CD surface antigens that are commonly used as phenotypic and functional markers in the mouse system. There are many reagents recognizing polymorphic epitopes of the mouse MHC and different subunits of TCR; for simplicity, only a few mAbs to the framework determinants of these molecules are listed here.

Reference: CD Antigens 2002.

The Proceedings of the 7th Human Leukocyte Differentiation Antigen (HLDA) Workshop are about to be published, detailing more than 80 new CD specificities. The next Workshop, planned for 2004, will continue this process, and a number of candidate CD molecules in the literature, identified by antibody

CD antigens 2001

This paper reviews the Seventh Human Leucocyte Differentiation Antigen (HLDA7) workshop. Due to the limitations of "blind" antibody screening, which had been evident at the previous meeting in 1996, participants at HLDA7 adopted a more selective approach to the choice of antibodies by identifying new CD specificities. This resulted in the addition of more than 80 new CD specificities. Plans for the eighth and subsequent workshops are also previewed.

CD antigens 2001.

CD antigens 2001.

CD antigens 2001.

The most recent Human Leucocyte Differentiation Antigen Workshop (HLDA7) took place in 2000 in Harrogate, UK and the proceedings are about to be published (Leucocyte Typing VII). New Sections were introduced in this Workship (Dendritic cells, Stem/progenitor cells, Erythroid cells and Carbohydrate Structures) and monoclonal antibodies were selected for which at least some molecular data were already available (to avoid blind screening of reagents against known specificities). A total of more than 80 new specificities were established (previously the average was less than 30 new specificities per Workshop) and these are listed in this article. There is already evidence for the existence of many new leucocyte surface molecules for study at the next HLDA Workshop (in Adelaide in 2004), and we have listed in this article a number of such potential candidates (identified following the production of monoclonal antibodies or via gene cloning). There are also today an increasing number of lineage- and/or stage-restricted leucocyte-associated molecules localised within the cell cytoplasm (or nucleus): they will certainly prove of intense in the future for many laboratories studying human haematopoietic cells (regardless of whether a new intracellular CD categorisation scheme is devised for such molecules).

CD antigens 2001.

The most recent Human Leucocyte Differentiation Antigen Workshop (HLDA7) took place in 2000 in Harrogate, UK and the proceedings are about to be published (Leucocyte Typing VII). New Sections were introduced in this Workship (Dendritic cells, Stem/progenitor cells, Erythroid cells and Carbohydrate Structures) and monoclonal antibodies were selected for which at least some molecular data were already available (to avoid blind screening of reagents against known specificities). A total of more than 80 new specificities were established (previously the average was less than 30 new specificities per Workshop) and these are listed in this article. There is already evidence for the existence of many new leucocyte surface molecules for study at the next HLDA Workshop (in Adelaide in 2004), and we have listed in this article a number of such potential candidates (identified following the production of monoclonal antibodies or via gene cloning). There are also today an increasing number of lineage- and/or stage-restricted leucocyte-associated molecules localised within the cell cytoplasm (or nucleus): they will certainly prove of intense in the future for many laboratories studying human haematopoietic cells (regardless of whether a new intracellular CD categorisation scheme is devised for such molecules).

CD Antigens 2001

The most recent Human Leucocyte Differentiation Antigen Workshop (HLDA7) took place in 2000 in Harrogate, UK and the proceedings are about to be published (Leucocyte Typing VII). New Sections were introduced in this Workship (Dendritic cells, Stem/progenitor cells, Erythroid cells and Carbohydrate Structures) and monoclonal antibodies were selected for which at least some molecular data were already available (to avoid blind screening of reagents against known specificities). A total of more than 80 new specificities were established (previously the average was less than 30 new specificities per Workshop) and these are listed in this article. There is already evidence for the existence of many new leucocyte surface molecules for study at the next HLDA Workshop (in Adelaide in 2004), and we have listed in this article a number of such potential candidates (identified following the production of monoclonal antibodies or via gene cloning). There are also today an increasing number of lineage- and/or stage-restricted leucocyte-associated molecules localised within the cell cytoplasm (or nucleus): they will certainly prove of intense in the future for many laboratories studying human haematopoietic cells (regardless of whether a new intracellular CD categorisation scheme is devised for such molecules).

Overview of the Third International Workshop on Swine Leukocyte Differentiation Antigens

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with ‘w’. As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).

Monoclonal antibodies raised to human cells — specificity for pig leukocytes

A total of 27 monoclonal antibodies raised to human targets were included in the present Pig CD workshop. 14 of these had been tested in previous workshops and had been reported as cross-reactive, a further 13 had been reported as cross-reactive during the Human Leukocyte Differentiation Antigens Workshop VI (HLDA VI) and/or by the donor (a commercial company submitting these mAb for validation by the workshop community). Of the 27 antibodies, three antibodies with previously reported reactivity for pig cells were eliminated from the workshop following preliminary tests due to lack of reactivity. Nine antibodies, although initially positive, gave inconsistent results during the course of the workshop. We found consistent reactivity for 15 antibodies. However, the cellular distribution of the target molecules on pig and human cells was shown to be different for three of these antibodies. These findings have important implications for the usefulness of these antibodies as research tools in the pig.

Summary of the first round analyses of the Third International Workshop on Swine Leukocyte Differentiation Antigens

The reactivity of 155 monoclonal antibodies submitted to the Third International Workshop on Swine Leukocyte Differentiation Antigens, together with 41 internal standards, was analysed by flow cytometry on 29 different pig cell targets as well as two human cell targets as a means of establishing suitable panels of monoclonal antibodies for more detailed clustering analyses by the various subsections of the workshop. Results were collected either without further gating, with gating based on FS/SS characteristics or with gating based on the co-expression of a reference antibody in two-colour flow cytometry. The CD or SWC reactivity of the internal standards had been established in previous workshops. Data sets were subsequently analysed by statistical clustering using the Leucocyte Typing Database IV software. The resulting 18 cluster groups were allocated to the appropriate second round sections of the workshop, after reviewing the overall cellular reactivity of each cluster as well as the specificity of known standards which clustered in a group.

Immunostimulatory effects of anionic alkali mineral complex solution Barodon in porcine lymphocytes

The anionic alkali mineral complex solution, Barodon (Barodon-S.F. Corp., Korea), was evaluated for its effectiveness as a nonspecific immunostimulator in pigs. The effects of Barodon were determined by analysis of feed efficiency, growth rate, and phenotype of leukocyte subpopulations using monoclonal antibodies specific to porcine leukocyte differentiation antigens and flow cytometry (FC). The study was focused to investigate the change in proportion of the CD4+CD8+ double positive T lymphocyte subpopulation (dpp) which exists uniquely in pigs. In addition, the mitogen-stimulated lymphoproliferative response, tissue distribution in lymphoid organs and the adjuvant effect of Barodon on hog cholera vaccine efficiency were determined. The study has revealed the average daily gain rates and feed conversion rates were significantly (p<0.05) improved in either group of pigs fed with 0.05% Barodon-spray feed (Tx-1) or pigs fed with 3% Barodon-fermented feed (Tx-2) in comparison with group of pigs fed with feed containing no Barodon (control). The proportion of cells expressing CD4+ antigen in Barodon-treated group increased from 3 weeks posttreatment and was significantly higher (p<0.05) than that of control at 8 weeks posttreatment. Particularly, the significantly higher proportion was maintained from 8 weeks through 13 weeks posttreatment in Tx-1 group (p<0.05). The proportion of cells expressing CD8+ antigen was significantly higher at 3 weeks posttreatment in Tx-2 (p<0.01). Proportion of MHC class II-expressing cells was significantly higher in Tx-1 and Tx-2 group at 11 weeks and 8 weeks posttreatment (p<0.05), respectively. In addition, the proportion of Non T/Non B (N) cells was also significantly higher in Tx-2 at 3 weeks posttreatment (p<0.01) and maintained to 13 weeks posttreatment (p<0.1). Between Barodon-treated groups, the proportion of MHC class II-expressing cells was observed to be larger in Tx-2 than Tx-1 from 3 weeks to 8 weeks posttreatment (p<0.05). However, there were no significant difference in the proportions of CD2+ cells, B cells, monocytes and granulocytes between Barodon-treated and control group during the experiment. Dual-color FC analysis, study has revealed an increased proportion of dpp present in lymphocytes obtained from peripheral blood (PB) and mesenteric lymph node (MLN) of Barodon-treated group at 8 and 11 weeks posttreatment. The proportion of dpp in PB was 27.5% and 32.1% in Tx-1 and Tx-2, respectively, but only 2.2% in control group at 8 weeks posttreatment. In MLN, the proportion was 45.1% and 52.1% in Tx-1 and Tx-2, respectively, otherwise 16.5% in control group at 8 weeks posttreatment. The mitogen-stimulated activity was significantly higher in Tx-1 than in the control group at 11 weeks posttreatment when cells were stimulated with Con A and PHA, respectively (p<0.01). Also, Con A-, PHA and PWM-stimulated activity was significantly higher in Tx-2 than in the control group at the same time (p<0.05). The tissue distribution of CD4+, CD8+ and CD4+CD8+ dpp in MLN and spleen was significantly larger in Tx-1 and Tx-2 than in the control group (p<0.01). Also, a larger proportion of dpp was observed in Tx-2 than Tx-1 in spleen between Barodon-treated groups (p<0.01). In conclusion, the study has demonstrated that Barodon had an immunostimulatory effect on pigs through proliferation and activation of porcine immune cells, specially CD4+CD8+ dpp lymphocytes.

Cross-reactivity with bovine cells of monoclonal antibodies submitted to the 6th International Workshop on Human Leukocyte Differentiation Antigens

Twelve subpanels of monoclonal antibodies (MAb) included within the 6th International Workshop on Human Leukocyte Differentiation Antigens (6th HLDA) were assayed for reactivity with bovine peripheral blood leukocytes. Sixty-nine of the 807 MAb (8.6%) stained bovine cells. These MAb represented 30 different human CD groups. Nine of the MAb to different human CD antigens (CD19, CD23, CD39, CD47, CD86, CD117, CD120b, CDw149, CD165) potentially recognized antigens on cattle cells that had not previously been identified. These were investigated further by two-colour immunofluorescence to compare the cellular expression of the antigen on cattle cells with that reported for the different CD antigens in humans. Four of the MAb that belonged to CD23, CD39, CD47, and CDw149 stained bovine cells in a manner that indicated an almost identical cellular distribution of the antigen to that reported in humans. This implied that these MAb reacted with the homologous cattle molecules. Further work would be necessary to confirm specificity of CD19, CD86, CD117, CD120b and CD165 MAb. Other cross-reacting MAb either recognized antigens already defined in cattle or antigens not yet clustered in humans. The study has identified valuable new reagents for studies of cattle and confirmed that most common cross-reactive MAb are to epitopes on integrins.

Preparation and characterization of mabs against different epitopes of CD226 (PTA1).

Recently the platelet and T-cell activation antigen 1 (PTA1) was assigned as CD226 at the 7th Conference and Workshop on Human Leukocyte Differentiation antigens (HLDA). PTA1 is mainly expressed on activated T cells, natural killer (NK) cells, platelets and stimulated endotheliocytes, and involved in the differentiation of cytotoxic T lymphocytes (CTL) and NK, as well as platelet activation and aggregation. We raised hybridomas secreting monoclonal antibodies (MAbs) to PTA1 by using the natural PTA1 as immunogen, which was purified from platelets via affinity chromatography. These MAbs, designated FMU1, FMU2, FMU3, FMU4, FMU5, FMU6 and FMU7, could recognize PTA1 cDNA transfected COS7 cells detected by flow cytometry (FCM), and also react with both natural PTA1 and PTA1/Ig fusion protein in indirect enzyme-linked immunoadsorbent assay (ELISA). The biosensor epitope mapping assay showed that the seven MAbs, together with previous PTA1-specific MAbs Leo A1 and New E1, could bind seven distinct epitopes of PTA1, respectively. The panel of MAbs might be new powerful tools to study the structure-function relationship of PTA1 molecule, and to search for the ligand of PTA1.

Immunophénotypage : Apport de la cytométrie à la biologie clinique

The interest of immunophenotyping by flow cytometry in clinical biology involves many fields : • description of normal blood cells, bone marrow cells and cells from various organs, • analysis of human leucocyte differentiation antigens, • studies of stem cells, cell birth, viability and programmed cell death, • description of cell signalling and functions, • the various immunopathology headings could be considered : immunodeficiencies, auto-immune diseases, transplantations, lympho-proliferative disorders, allergy, • application in the diagnosis of hematologic disorders, • cancer research, immunosurvey against tumor cells and immunotherapy. The ability to identify cellular antigens, and associate the studies of nucleic acid or cell ploidy by flow cytometry is a major step in understanding physiological mechanisms in many diseases.

Premature infants respond to early-onset and late-onset sepsis with leukocyte activation

Objective: Leukocyte differentiation antigens are expressed on the cell membrane during activation. The purpose of this study was to evaluate leukocyte activation in premature neonates with sepsis. Paired blood samples from the same individual while sick and while convalescent were examined to quantify the expression of leukocyte antigens in these clinical states. Methods: Mononuclear blood cells from 21 premature infants (24 to 30 weeks’ gestation) were analyzed. The “sick” samples were drawn at the time of workup for sepsis; “convalescent” samples were drawn 20 days later. Samples were incubated with monoclonal antibodies to the lymphocyte antigens CD3, CD19, CD25, CD26, CD71, and CD69 and neutrophil antigens CD11b, CD11c, CD13, CD15, CD33, and CD66b. The cells were lysed, fixed, and analyzed by flow cytometry. Results: Twenty-one infants enrolled in the study had multiple sepsis evaluations and had more than one sample available for a paired observation. CD33, CD66b, and CD19 levels were significantly elevated in both the presumed sepsis and culture-proven sepsis groups when compared with the samples drawn from those same patients when healthy. Expression of CD33 and expression of CD66b were correlated, and in a multivariate analysis the elevation of antigen expression was predictive of sepsis. Conclusions: Leukocytes from preterm newborn infants respond to infection with an increased expression of CD19, CD33, and CD66b on their cell surfaces. (J Pediatr 2000;137:345-50)

CDw149 antibodies recognize a clustered subset of CD47 molecules associated with cytoplasmic signaling molecules.

One of the recently described antigens broadly expressed on human leukocytes is CDw149, which was defined at the 6th Human Leukocyte Differentiation Antigen (HLDA) Workshop by means of 2 monoclonal antibodies (mAbs). Molecular characterization of this antigen has been lacking. In the present study we demonstrate that these anti-CDw149 mAbs actually recognize a clustered subset of a well-defined membrane protein, CD47, also known as integrin-associated protein (IAP). This clustered subset is present on leukocytes but not erythrocytes. The anti-CDw149 mAbs bind with only low affinity to a monomeric (unclustered) subset of CD47 but with high avidity to the CD47 clusters. A fraction of CD47 is associated with large complexes containing cytoplasmic signaling molecules (Src family kinases and heterotrimeric G-proteins) similar to glycosphingolipid-enriched microdomains (GEMs), which may explain the previously described signaling capacity of CD47. The low-affinity anti-CD47 mAbs may be useful tools targeting specific receptor complexes involved in cell activation. Specific reactivity of low-affinity mAbs with clustered subsets of cell surface antigens may more generally explain the nature of poorly defined "activation forms" or activation neoepitopes described previously for several cell surface molecules.

Predominant subpopulations of T lymphocytes in the mammary gland secretions during lactation and intraepithelial T lymphocytes in the intestine of dairy cows

Lymphocytes obtained from mammary gland secretions (MGS) during lactation or the dry period of dairy cows were simultaneously analyzed and compared to ileal intraepithelial lymphocytes (IEL) and peripheral blood lymphocytes (PBL) using monoclonal antibodies (mAb) specific for bovine leukocyte differentiation antigens. The T-lymphocytes of MGS during lactation and those in IEL were predominantly CD8 +, while T-cells in MGS during the dry period were predominantly CD4 +. In addition, the proportion of γ δ T-cells in MGS during lactation and IEL was fairly high. A large percentage of CD8 + cells and T-cells coexpressed the activation molecule, ACT2, yielding a high proportion of ACT2 + CD8 T-cells and ACT2 + γ δ T-cells, in MGS during lactation and IEL. However, both types of cells were found at an extremely low level in MGS during the dry period and in PBL. Thus, the predominant T-cell populations in MGS during lactation are phenotypically similar to those in IEL in the intestine.

Elevated cyclin E levels, inactive retinoblastoma protein, and suppression of the p27(KIP1) inhibitor characterize early development of promyeloid cells into macrophages.

Cyclin-dependent kinase inhibitors such as p27(KIP1) have recently been shown to lead to cellular differentiation by causing cell cycle arrest, but it is unknown whether similar events occur in differentiating promyeloid cells. Hematopoietic progenitor cells undergo lineage-restricted differentiation, which is accompanied by expression of distinct maturation markers. Here we show that the classical growth factor insulin-like growth factor I (IGF-I) potently promotes vitamin D(3)-induced macrophage differentiation of promyeloid cells, as assessed by measurement of a coordinate increase in expression of the integrin alpha subunit CD11b, the CD14 lipopolysaccharide receptor, and the macrophage-specific esterase, alpha-naphthyl acetate esterase, as early as 24 h following initiation of terminal differentiation. Addition of IGF-I to cells undergoing vitamin D(3)-induced differentiation also leads to an early increase in expression of cyclin E, phosphorylation of the retinoblastoma tumor suppressor protein, and a doubling of the cell number. Early expression of CD11b (24 h) is simultaneously accompanied by inhibition in the expression of p27(KIP1). Cell cycle analysis with propidium iodide revealed that CD11b expression at 24 h following initiation of differentiation occurs at all phases of the cell cycle instead of only those cells arrested in G(0)/G(1). Similarly, development of a novel double-labeling intra- and extracellular flow-cytometric technique demonstrated that single cells expressing the mature leukocyte differentiation antigen CD11b can also incorporate the thymidine analog bromodeoxyuridine. Likewise, expression of the intracellular DNA polymerase delta cofactor/proliferating-cell nuclear antigen at 24 h is also simultaneously expressed with the surface marker CD11b, indicating that these cells continue to proliferate early in their differentiation program. Finally, at 24 h following induction of differentiation, IGF-I promoted a fourfold increase in the uptake of [(3)H]thymidine by purified populations of CD11b-expressing cells. Taken together, these data demonstrate that the initial steps associated with terminal macrophage differentiation occur concomitantly with progression through the cell cycle and that these very early differentiation events do not require the accumulation of p27(KIP1).

Characterization of porcine T lymphocytes and their immune response against viral antigens

T lymphocytes play a central role in the antigen-specific immune response against various pathogens. To detect and to characterize porcine T lymphocytes, monoclonal antibodies (mAb) against leukocyte differentiation antigens had been raised and classified for their specificity. Analyses of porcine T lymphocytes with specific mAb against CD4 and CD8 differentiation antigens revealed differences in the composition of the porcine T-lymphocyte population compared to other species. In addition to the known subpopulations, CD4 +CD8 − T helper cells and CD4 −CD8 + cytolytic T lymphocytes, extra-thymic CD4 +CD8 + T lymphocytes and a substantial proportion of CD2 −CD4 −CD8 − T cell receptor (TcR)-γδ + T cells could be detected in swine. Functional analyses of porcine T-lymphocyte subpopulations revealed the existence of two T-helper cell fractions with the phenotype CD4 +CD8 − and CD4 +CD8 +. Both were reactive in primary immune responses in vitro, whereas only cells derived from the CD4 +CD8 + T-helper-cell subpopulation were able to respond to recall antigen in a secondary immune response. With regard to T lymphocytes with cytolytic activities, two subsets within the CD4 −CD8 + T-cell subpopulation could be defined by the expression of CD6 differentiation antigens: CD6 − cells which showed spontaneous cytolytic activity and CD6 + MHC I-restricted cytolytic T lymphocytes including virus-specific cytolytic T lymphocytes. These results enable now a detailed view into the porcine T-cell population and the reactivity of specific T cells involved in the porcine immune response against pathogens. Furthermore this knowledge offers the possibility to investigate specific interactions of porcine T lymphocytes with virus-specific epitopes during vaccination and viral infections.

Inhibition of dipeptidyl peptidase IV by fluoroolefin-containing N-peptidyl-O-hydroxylamine peptidomimetics.

Dipeptidyl peptidase IV (EC 3.4.14.5; DPP IV), also known as the leukocyte differentiation antigen CD26 when found as an extracellular membrane-bound proline specific serine protease, cleaves a dipeptide from the N terminus of a polypeptide chain containing a proline residue in the penultimate position. Here we report that known (Z)-Ala-psi[CF=C]-Pro dipeptide isosteres 1 and 2, which contain O-acylhydroxylamines, were isolated as diastereomeric pairs u-1, l-1, and l-2. The effect of each diastereomeric pair as an inhibitor of human placental dipeptidyl peptidase DPP IV has been examined. The inhibition of DPP IV by these compounds is rapid and efficient. The diastereomeric pair u-1 exhibits very potent inhibitory activity with a Ki of 188 nM. Fluoroolefin containing N-peptidyl-O-hydroxylamine peptidomimetics, by virtue of their inhibitory potency and stability, are superior to N-peptidyl-O-hydroxylamine inhibitors derived from an Ala-Pro dipeptide.