Human Leukocyte Differentiation Antigens Research Articles

Integrated Network Pharmacology and Clinical Study to Reveal the Effects and Mechanisms of Bushen Huoxue Huatan Decoction on Polycystic Ovary Syndrome.

Objective Bushen Huoxue Huatan Decoction (BHHD) is a classic prescription for treating polycystic ovary syndrome (PCOS). This study aims to explore the effects and possible mechanisms of BHHD on PCOS by integrating network pharmacology and clinical study. Methods The components and potential drug targets of BHHD were analysed using the TCMSP platform, and the potential pathogenesis targets for PCOS were analysed using the GeneCards and OMIM databases. Subsequently, a disease-compound-target network diagram was established to identify the targets of BHHD treatment on PCOS. In addition, protein-protein interaction analysis, KEGG pathway analysis, and Gene Ontology biological analysis were carried out to reveal the mechanisms of BHHD. To further validate the analysis, a clinical trial involving 62 PCOS patients was conducted. All patients were treated with BHHD for 3 months and the ovulation rate, anthropometric indicators, clinical symptoms, and serological indicators were measured and compared before and after treatment. Results The network pharmacology analysis showed that quercetin, luteolin, and kaempferol are the most significant active components in BHHD; STAT3, Jun, AKT1, MAPK3, MAPK1, and TP53 are the most critical drug targets; regulating hormones, reversing insulin (INS) resistance, exerting anti-inflammatory effects, and improving fertility might be the most important mechanisms of BHHD in the treatment of PCOS. After BHHD administration, the cyclic ovulation rate and the clinical symptoms including acanthosis nigricans and acne of patients were obviously improved. The serum endocrine levels of luteinising hormone (LH), LH/follicle-stimulating hormone, testosterone, dehydroepiandrosterone sulphate, insulin (INS), and area under the INS curve were evidently reversed, and the serum inflammatory factors levels including human interleukin (IL)-18, IL-16, IL-1β, IL-8, macrophage migration inhibitory factor, and human leukocyte differentiation antigen CD40 ligand were greatly reduced. Conclusion BHHD has a good therapeutic effect on PCOS, and its mechanisms may be related to regulating hormone levels, improving insulin resistance, alleviating inflammation, and promoting pregnancy.

Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative.

BackgroundThe Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking.ObjectiveTo develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories.MethodsA high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ+, regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation.ResultsUsing automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen.ConclusionWe optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops.

Genome-wide polygenic analysis of multiple sclerosis markers

Objective: to perform a genome-wide polygenic analysis of multiple sclerosis (MS) markers in the ethnic groups of Bashkirs, Russians, and Tatars living in the Republic of Bashkortostan (Russian Federation).Patients and methods. Genotyping was performed using allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism analysis of genes of the human leukocyte differentiation antigens CD6 (rs17824933), CD40 (rs6074022), CD58 (rs2300747), CD86 (rs9282641), transcription factors SOX8 (rs2744148) and ZBTB46 (rs6062314), beta-mannosidase MANBA (rs228614), C-type lectin domain CLEC16A (rs12708716), ribosomal protein S6 kinase B1 RPS6KB1 (rs180515), and long noncoding RNA gene PVT1 (rs759648) in 644 patients with MS and 1408 controls. Multilocus analysis of the disease associations with combinations of genotypes and alleles of the studied polymorphic loci was performed using the APSampler algorithm.Results and discussion. We determined the distribution of genotype and allele frequencies of the studied polymorphic loci in the ethnic groups of Bashkirs, Russians, and Tatars. We also observed disease associations with CD58 (rs2300747) and RPS6KB1 (rs180515) polymorphic loci in Russian men, CD86 (rs9282641) in Russian, PVT1 (rs759648) in Tatar women, CD40 (rs6074022) in Bashkir men, and identified 19 combinations of genotypes and/or alleles significantly associated with MS.Conclusion. Based on the genome-wide polygenic analysis of MS markers, we identified ethno- and gender-specific combined markers of the disease susceptibility.

Correlation analysis between the progression of HIV infection and HLA polymorphism

Objective: To analyze the polymorphisms of human leukocyte differentiation antigens Ⅰ and Ⅱ (HLA-A, B, DRB1) alleles and explore the association between HIV infection and HLA loci, for discovering the distribution of HLA loci in HIV-infected with different disease progression in different parts of Henan Province. Methods: A total of 48 cases of slow progressers and 80 typical progressers in Weishi County, Shangcai County, Xihua County and Xuchang City of Henan Province were studied, and compared with 380 healthy blood donors.For analyzing HLA-A, B, DRB1 alleles and comparing difference among the study subjects, the method of polymerase chain reaction-sequence special oligonucleotide (PCR-SSO) was used. Results: The association of HLA alleles and HIV infection showed that HLA-B*40∶02, HLA-DRB1*04∶05 was significantly more common in healthy people, while HLA-B*15∶18, B*44∶02, B*67∶01 and HLA-DRB1*14∶01 were present in HIV/AIDS.HLA-A*02∶06, HLA-B*13∶02, B*40∶06 in slow progressers were higher than typical progressers from the grouped study, and HLA-B*46∶01 only appeared in the typical progressers. Conclusion: HLA-B*15∶18, B*44∶02, B*67∶01, and HLA-DRB1*14∶01 may be associated with HIV susceptibility.HLA-A*02∶06, HLA-B*13∶02, and B*40∶06 may be associated with delayed disease progression in HIV-infected people, while HLA-B*46∶01 may be associated with accelerated disease progression.

Improvement of clinical immunofixation electrophoresis assay for C3 nephritic factor

Human cell differentiation molecules is a new terminology coined by the 8th International Workshop on Human Leukocyte Differentiation Antigens in 2004 to describe surface molecules associated with human cell differentiation. These molecules have been characterized in a series of international workshops studying a large number of monoclonal antibodies. The antibodies have been grouped according to their patterns of reactivity and are referred to as “clusters of differentiation (CD).”

Universal cell type identifier based on number theory.

Cell type classification and handling is a key issue for understanding biological systems. The advent of high multiplexing technologies increased the complexity of the classification process and new tools are needed to support the organization of this knowledge. Ipropose a classification based on both prime numbers and the fundamental theorem of arithmetic. As a not limiting example, I show the application of this method to unambiguously define any existing cell type using the CD nomenclature established by the Human Leukocyte Differentiation Antigens Workshops. This system allows for the unique identification of any possible combination of markers hence any cell population without previous knowledge and without the need to increment the system. This method can be the future basis of any database and ontology system dealing with cell types and beyond the biological field applies to the description of any entity characterized by a list of discrete qualities. © 2018 International Society for Advancement of Cytometry.

Determination of normal expression patterns of CD86, CD210a, CD261, CD262, CD264, CD358, and CD361 in peripheral blood and bone marrow cells by flow cytometry

In 2010, new monoclonal antibodies were submitted to the 9th International Workshop on Human Leukocyte Differentiation Antigens, and there are few studies demonstrating normal expression patterns of these markers. Thus, the objective of this study was to determine the normal patterns of cell expression of CD86, CD210a, CD261, CD262, CD264, CD358, and CD361 in peripheral blood (PB) and bone marrow (BM) samples by flow cytometry. In the present study, CD86 was expressed only in monocytes and B lymphocytes in PB and in monocytes and plasma cells in BM. Regarding CD210a expression, in PB samples, monocytes and NK cells showed weak expression, while neutrophils, B and T lymphocytes, and basophils showed weak and partial expression. In BM samples, expression of CD210a was observed in eosinophils, monocytes, and B and T/NK lymphocytes. Weak expression of CD210a was also observed in neutrophilic cells and plasma cells. All B cell maturation stages had weak expression of CD210a except for immature B cells, which did not express this marker. In the present study, no cell type in PB samples showed positivity for CD261 and, in BM samples, there was very weak expression in neutrophilic series, monocytes, and B lymphocytes. Conversely, plasma cells showed positivity for CD261 with a homogeneous expression. For CD262, there was weak expression in monocytes, neutrophils, and B lymphocytes in PB samples and weak expression in monocytes, B lymphocytes, and plasma cells in BM samples. The evaluation of CD264 showed very weak expression in B cells in PB samples and no expression in BM cells. Very weak expression of CD358 was observed in neutrophils, monocytes, and B lymphocytes in PB and BM samples. In addition, in BM samples, plasma cells and T lymphocytes showed weak expression of CD358. In relation to the maturation stages of B cells, there was weak expression in pro-B cel, pre-B cell, and mature B cell. In the present study, it was possible to observe expression of CD361 in all cell types analyzed in PB and BM samples. The analyzed markers presented varied profiles of expression and, in some cases, these profiles were different from those observed in other studies. Further studies are needed to evaluate these molecules, mainly in relation to a possible application in the diagnosis of hematological malignancies or as new therapeutic targets for the treatment of hematological neoplasms or autoimmune diseases.

In silico identification of cytotoxic T lymphocyte epitopes encoded by RD5 region of Mycobacterium tuberculosis.

While CD8+ T cells (cytotoxic T lymphocytes, CTLs) play important roles in immunity against Mycobacterium tuberculosis, only a small number of human leukocyte differentiation antigen (HLA) class I-restricted CTL epitopes for TB have been identified. The current study evaluates CTL epitopes of Rv3117 and Rv3120 proteins, two newly found M. tuberculosis region-diffference-5 (RD5)-encoded antigens, and their population coverage. The amino acid sequences of the two proteins were subjected to epitope analysis under HLA-A2, A3 and B7 supertype restriction using NetCTL, SYFPEITHI, BIMAS, NetCTLPan, IEDB, NetMHC and NetMHCPan prediction online servers. Eight RD5-encoded CTL epitopes were identified in the two proteins and the average population coverage of these epitopes was 87.2% among populations worldwide. These CTL epitopes that were identified in silico and may have potential use for CD8+ T cell-mediated TB vaccine design.

CD16+ Dendritic Cells Are a Unique Myeloid Antigen Presenting Cell Population

Dendritic cells (DC) are phenotypically identified in human blood as HLA-DR+ cells, which lack major cell surface lineage markers. We demonstrated that myeloid antigen presenting cells, including monocytes and DC display a continuum of CD14 and CD16 expression (10th Human Leucocyte Differentiation Antigen Workshop). The robustness of DC and monocyte identification, particularly when identifying cell subsets with little or no surface CD14 is limited by subjective gating strategies for determining rare cell populations. Application of Poisson counting statistics established that rare cell types such as "CD14- CD16+ DC" are often overlooked in analyses powered to detect the much larger populations of "classical" and "non-classical" monocytes.We used fluorescent and mass cytometry, in conjunction with unsupervised high dimensional clustering, to show that the continuum of CD14 expression separates CD14lo CD16+ non-classical monocytes and CD14- CD16+ DC. We have defined the CD14-CD16+ DC using a broad panel of cell surface markers and established a CD14-CD16+ DC phenotypic signature that is distinct from both classical and non-classical monocytes in healthy donor blood. The CD14-CD16+ DC differ in both size to CD14lo CD16+ monocytes and functional antigen uptake, with slower kinetics of soluble antigen uptake into lysozymes. Their proteasome processing and presentation of influenza matrix protein by MHC I was comparable to other primary blood monocytes and DC antigen presenting cell populations. The CD14-CD16+ DC had limited capacity for further in vitro differentiation. The recovery of CD14-CD16+ DC after autologous and allogeneic myeloablative hematopoietic cell transplants (HCT) followed similar kinetics to other monocytic and DC populations. CD14lo CD16+ monocytes expressed CCR5 as did other myeloid DC but CD14-CD16+ DC lacked CCR5, although interferon induced CCR5. The early differentiation and induction of CCR5 on circulating CD16+ DC after allogeneic HCT predicted for the onset of acute graft versus host disease.These data demonstrate that "CD14- CD16+ DC" represents a distinct clinically relevant human white blood cell population, whose ontogeny and function are under further investigation. DisclosuresFromm:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Papadimitrious:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Hsu:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Larsen:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Gibson:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Bradstock:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Kupresanin:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Clark:DendroCyte BioTech Ltd: Equity Ownership, Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Hart:DendroCyte BioTech Ltd: Equity Ownership, Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd.

Expression profile of novel cell surface molecules on different subsets of human peripheral blood antigen‐presenting cells

Although major steps have been recently made in understanding the role of the distinct subsets of dendritic cells (DC)/antigen-presenting cells (APC), further studies are required to unravel their precise role, including in-depth immunophenotypic characterisation of these cells. Here, we used eight-colour flow cytometry to investigate the reactivity of a panel of 72 monoclonal antibodies (including those clustered in seven new Cluster of Differentiation, CD) on different subsets of APC in peripheral blood (PB) samples from five healthy adults. These experiments were performed in the context of the Tenth International Workshop on Human Leukocyte Differentiation Antigens (HLDA10). Plasmacytoid DC was the only cell population that expressed CD85g and CD195, whereas they lacked all of the other molecules investigated. In contrast, myeloid DC mostly expressed inhibitory C-type lectin receptors (CLRs) and other inhibitory-associated molecules, whereas monocytes expressed both inhibitory and activating CLRs, together with other phagocytosis-associated receptors. Within monocytes, progressively lower levels of expression were generally observed from classical monocytes (cMo) to SLAN− and SLAN+ non-classical monocytes (ncMo) for most of the molecules expressed, except for the CD368 endocytic receptor. This molecule was found to be positive only in cMo, and the CD369 and CD371 modulating/signalling receptors. In addition, the CD101 inhibitory molecule was found to be expressed at higher levels in SLAN+ vs SLAN− ncMo. In summary, the pattern of expression of the different signalling molecules and receptors analysed in this work varies among the distinct subsets of PB APCs, with similar profiles for molecules within each functional group. These findings suggest unique pattern-recognition and signalling capabilities for distinct subpopulations of APCs, and therefore, diverse functional roles.

Nomenclature of CD molecules from the Tenth Human Leucocyte Differentiation Antigen Workshop.

Kohler and Milstein’s1 description of monoclonal antibodies (mAbs) in 1975 revolutionised immunology. With the advent of hybridoma technology, immunologists started to produce an ever increasing number of mAbs directed against leukocyte cell-surface molecules. Inevitably, several mAbs produced by different laboratories and given independent names recognised the same molecule. The First International Human Leucocyte Differentiation Antigen (HLDA) Workshop was organised in the early 1980s, and aimed to identify groups of mAbs reacting with a common human cell-surface antigen and to agree a nomenclature that would facilitate better communication amongst the scientific community.2 The outcomes of the Workshops are the ‘CDs’, an abbreviation for the non-descriptive ‘cluster of differentiation’ number. The CD number is assigned to a group or cluster of mAbs that recognise a molecule expressed on the surface of human leukocytes and other cells relevant to the immune system. The nomenclature has been universally adopted by the scientific community, officially approved by the International Union of Immunological Societies and sanctioned by WHO. Its usage has now evolved and is also commonly used to name the molecules themselves. However, by adding either mAb or molecule/protein it should be made clear whether one means the CDxx mAb or CDxx molecule. Ten HLDA Workshops have been organised to date, with the most recent one held in 2014 in conjunction with the Australasian Society of Immunology in Wollongong, Australia.

New insights into the phenotype of human dendritic cell populations.

HLDA10 is the Tenth Human Leukocyte Differentiation Antigen (HLDA) Workshop. The HLDA Workshops provide a mechanism to allocate cluster of differentiation (CD) nomenclature by engaging in interlaboratory studies. As the host laboratory, we invited researchers from national and international academic and commercial institutions to submit monoclonal antibodies (mAbs) to human leukocyte surface membrane molecules, particularly those that recognised molecules on human myeloid cell populations and dendritic cells (DCs). These mAbs were tested for activity and then distributed as a blinded panel to 15 international laboratories to test on different leukocyte populations. These populations included blood DCs, skin-derived DCs, tonsil leukocytes, monocyte-derived DCs, CD34-derived DCs, macrophage populations and diagnostic acute myeloid leukaemia and lymphoma samples. Each laboratory was provided with enough mAb to perform five repeat experiments. Here, we summarise the reactivity of different mAb to 68 different cell-surface molecules expressed by human myeloid and DC populations. Submitted mAbs to some of the molecules were further validated to collate data required to designate a formal CD number. This collaborative process provides the broader scientific community with an invaluable data set validating mAbs to leukocyte-surface molecules.

Differentiation of human monocytes and derived subsets of macrophages and dendritic cells by the HLDA10 monoclonal antibody panel.

The mononuclear phagocyte system, consisting of monocytes, macrophages and dendritic cells (DCs), has an important role in tissue homeostasis as well as in eliciting immune responses against invading pathogens. Blood monocytes have been viewed for decades as precursors of tissue macrophages. Although the newest data show that in the steady state resident macrophages of many organs are monocyte independent, blood monocytes critically contribute to tissue macrophage and DC pools upon inflammation. To better understand the relationship between these populations and their phenotype, we isolated and differentiated human blood CD14+ monocytes in vitro into immature and mature monocyte-derived dendritic cells (MoDCs) as well as into seven different monocyte-derived macrophage subsets. We used the panel of 70 monoclonal antibodies (mAbs) submitted to the 10th Human Leukocyte Differentiation Antigen Workshop to determine the expression profiles of these 10 populations by flow cytometry. We now can compile subpanels of mAbs to differentiate the 10 monocyte/macrophage/MoDC subsets, providing the basis for novel diagnostic and therapeutic tools.

CD Nomenclature 2015: Human Leukocyte Differentiation Antigen Workshops as a Driving Force in Immunology.

CD (cluster of differentiation) Ags are cell surface molecules expressed on leukocytes and other cells relevant for the immune system. CD nomenclature has been universally adopted by the scientific community and is officially approved by the International Union of Immunological Societies and sanctioned by the World Health Organization. It provides a unified designation system for mAbs, as well as for the cell surface molecules that they recognize. This nomenclature was established by the Human Leukocyte Differentiation Antigens Workshops. In addition to defining the CD nomenclature, these workshops have been instrumental in identifying and determining the expression and function of cell surface molecules. Over the past 30 y, the data generated by the 10 Human Leukocyte Differentiation Antigens Workshops have led to the characterization and formal designation of more than 400 molecules. CD molecules are commonly used as cell markers, allowing the identification and isolation of leukocyte populations, subsets, and differentiation stages. mAbs against these molecules have proven to be essential for biomedical research and diagnosis, as well as in biotechnology. More recently, they have been recognized as invaluable tools for the treatment of several malignancies and autoimmune diseases. In this article, we describe how the CD nomenclature was established, present the official updated list of CD molecules, and provide a rationale for their usefulness in the 21st century.

Construction and expression of a human/mouse chimeric CD19 monoclonal antibody: Successful modification of a murine IgM to a chimeric IgG

CD19 is a specific surface marker of B cells. A murine IgM-subtype antibody, 2E8, was generated previously and assigned to the CD19 category by the 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens in 1996. In the present study, the 2E8 Fv gene was inserted into a baculovirus shuttle vector and novel protein was expressed in an IgG1 form in the Sf9 insect cell line. VH2E8 and VL2E8 genes were cloned and inserted into the baculovirus shuttle vector pAc-κ-CH3 to form pAc-κ-CH3-VH2E8-VL2E8. Sf9 cells were then transfected with the reconstructed baculovirus shuttle vector. Novel protein expressed by the Sf9 cells was identified by immunofluorescence and western blot analysis, while activity levels were analyzed by flow cytometry (FCM). Sequencing demonstrated that the VH2E8 and VL2E8 fragments were inserted into pAc-κ-CH3 correctly. The immunofluorescence, western blot analysis and FCM results indicated that active recombinant antibody was expressed in the cytoplasm of Sf9 cells, but not in the culture supernatant. Thus, functional recombinant antibody was expressed successfully in the cytoplasm of Sf9 cells, but was not secreted into the culture supernatant. Therefore, the present study demonstrates that it is possible to modify mouse IgM to mouse-human chimeric IgG1 while retaining reasonable biological activity.

Sema 4D/CD100-plexin B is a multifunctional counter-receptor

The discovery of Sema 4D can be dated to 1992, when Boumsell's team reported a novel 150-kDa glycoprotein dimer. It was expressed on T lymphocytes that had been activated with CD3 monoclonal antibody or phytohaemagglutinin.1 Hence, this molecule has been designated CD100 by the International Workshop on Human Leukocyte Differentiation Antigen. In 1996, 3 years after the discovery of the semaphorin family,2 the CD100 gene was cloned and identified as the first semaphorin with immune system functions.3 Unlike the prototypical semaphorins, which are neuronal chemorepellents, Sema 4D/CD100 together with Sema 3A, Sema 4A, Sema 6D and Sema 7A have been described as ‘immune semaphorins', because they are expressed on T cells, B cells, natural killer cells and dendritic cells, and exhibit a variety of immunological functions.4 The Sema 4D/CD100 molecule can bind to several receptors. CD72, a member of the C-type lectin family, is a low-affinity Sema 4D/CD100 receptor that is expressed on immune cells, such as B cells, dendritic cells, macrophages and mast cells (Figure 1a).5 At least three Sema 4D/CD100 receptors are plexin family members. The high-affinity Sema 4D/CD100 receptor plexin B1 is mainly expressed on endothelial cells and epithelial cells (Figure 1b),6 whereas plexin B2 and plexin C1 are low-affinity receptors for Sema 4D/CD100.7 Figure 1 Multiple functions mediated by Sema 4D/CD100-plexin B counter-receptors. (a) As a ligand, the ligation of Sema 4D/CD100 with the low-affinity receptor CD72 stimulates the activation of, proliferation of and antibody production by B cells, promotes the ... Recently, Witherden et al.8 demonstrated that the interaction of Sema 4D/CD100 with plexin B2 plays a key role in activating skin-resident γδ T cells (DETCs) in mice to respond to tissue damage (Figure 1c). The morphological changes in DETCs that precede cutaneous wound healing appear to be associated with the activation of key actin regulators in response to Sema 4D/CD100's binding with its ligand. This is the first example indicating that Sema 4D/CD100 is not only a ligand for plexin B1 or CD72 but also a direct signaling receptor in mediating Sema 4D/CD100-plexin B's biological functions. It is not surprising to identify Sema 4D/CD100-plexin B as a counter-receptor, because many counter-receptors have already been well documented in the B7-CD28 family and TNF–TNFR superfamilies. Another important point in Witherden's paper8 is that the Sema 4D/CD100 molecule on γδ T cells mediates a function involved in innate immunity. Bonneville9 pointed out that plexin B2 is broadly expressed on many epithelial tissues where resident CD100-expressing γδ T cells are located, suggesting that the Sema 4D/CD100–plexin B2 interaction may play a more general role in the immune control of the integrity of epithelial barriers. It is thought that, similar to αβ T cells, complete activation of DETCs requires costimulatory signals. DETCs do not express many of the usual coreceptors that are important for αβ T-cell activation, such as CD4 or CD8, or the costimulatory molecule CD28. In 2010, Witherden et al.10 identified a receptor–ligand pair, junctional adhesion molecule-like molecule and the cocksackie adenovirus receptor (CAR), which plays a crucial role in the DETC response. In addition, DETCs can recognize a stress- or damage-induced keratinocyte self-antigen through their γδ T-cell receptor (TCR), without the requirement for antigen presentation by MHC class I or class II molecules. More recently, Komori et al.11 demonstrated the presence of an as yet-uncharacterized antigen that is expressed on the surface of damaged keratinocytes and is recognized by the canonical TCR expressed by DETCs. Furthermore, Witherden et al.8 proposed that when keratinocytes are damaged, they express a self-antigen, plexin B2 and CAR, which can be recognized by the γδ TCR, Sema 4D/CD100 and junctional adhesion molecule-like molecule, respectively, on neighboring γδ T cells (Figure 1c). Therefore, these interactions may play a key role in regulating the ERK and cofilin signaling pathways of activated γδ T cells, leading to morphologic changes and the release of keratinocyte growth factor type 1 (KGF1) and KGF2. These growth factors can induce the proliferation and migration of keratinocytes and the restoration of epithelial integrity.

Porcine SWC1 is CD52—Final determination by the use of a retroviral cDNA expression library

During the last decades for several species –e.g. swine – many mAb to leukocyte-specific molecules have been developed and clusters of differentiation corresponding to human CD could be established. However, for a significant amount of the raised mAb the corresponding antigens were not characterized on the molecular level and therefore preliminary clusters – in swine so-called Swine workshop clusters (SWC) – were established. These clusters contain antigens with currently no obvious orthologs to human leukocyte differentiation antigens. In this study, we describe the generation of a eukaryotic cDNA expression library from in vitro activated porcine peripheral blood mononuclear cells. Screening of this library with an antibody recognizing SWC1 enabled isolation and sequencing of cDNAs coding for the porcine SWC1 molecule. A BLAST search of the obtained sequence revealed that SWC1 is the orthologous molecule of human CD52. Therefore, our study provides the basis for comparative studies on the role of CD52 in different mammalian species. In addition, the established cDNA library can be used for investigation of additional SWC-defined molecules.

Expression profiles of novel cell surface molecules on B-cell subsets and plasma cells as analyzed by flow cytometry

Cell surface molecules are present on several lymphocyte subsets and are differentially expressed during lymphocyte development and activation. Human Leukocyte Differentiation Antigen (HLDA) Workshops have played an essential role in the identification and characterization of the molecules found in the membrane of hematopoietic cells. In the present study, the reactivities of sixty-five monoclonal antibodies (mAbs) submitted to the HLDA9 Workshop were tested. A multicolor flow cytometric analysis was performed in order to determine the expression profiles of these proteins on peripheral blood lymphocytes, hematopoietic cell lines, and tonsil B-cells. The following B-cell subsets were assessed: mature naïve, pre-germinal center, germinal center, unswitched and switched memory, plasmablasts, and plasma cells. Immunohistochemical analysis on formalin-fixed paraffin-embedded tonsils was also carried out. Remarkably, a large group of immunoglobulin family inhibitory cell surface molecules were observed on several distinct B-cell subsets including: CD152 (CTLA4), CD170 (Siglec-5), CD272 (BTLA), CD305 (LAIR1), CD307d (FCRL4), and CD329 (Siglec-9). The following molecules were also found to be differentially expressed on B-cell subsets (CD80, CD185 (CXCR5), CD196 (CCR6), CD270 (TNFRSF14), CD307a–c (FCRL1–3), CD319 (SLAMF7) and CD362 (SDC2)) or delineated B-cell subpopulations (CD126 (IL-6R), CD255 (TNFSF12), CD264 (TNFRSF10D), CD267 (TNFRSF13B) and CD329 (Siglec-9)). Of these, only CD307a, CD307b, and CD307d presented a B-cell-specific expression pattern. Our results show that several of these molecules are capable of further subdividing the known B-cell subsets and, in fact, may represent new markers for research, diagnosis, and eventually targets for the treatment of B-cell malignancies and autoimmune diseases.

New monoclonal antibodies against B-cell antigens: Possible new strategies for diagnosis of primary cutaneous B-cell lymphomas

Reactivities of the monoclonal antibodies (mAbs) of the 9th Human Leukocyte Differentiation Antigen Workshop, in order to define specific antigenic expression of the primary cutaneous B-cell lymphomas (PC-BCL), were analyzed by immunohistology on human tonsil and on PC-BCL, such as follicular centre B-cell lymphomas (FCL), marginal zone lymphomas (MZL) and diffuse large B-cells lymphomas leg-type (DLBL-LT). We identified some subgroups of mAbs that were exclusively or preferentially positive in one lymphoma cell type: the PC-FCL subgroup of mAbs includes PD1/CD279, GCET-1, hFCRL1/CD307a, FCRL2/CD307b, CXCR5/CD185, B7-DC/CD273, MRC/CD200, CD130, CXCR4/CD184, Siglec-5/14, CD150, on the other hand subgroup of mAbs in PC-MZL includes BTLA/CD272, BLIMP-1, hCD38. No specific subgroup of mAbs was found to label PC-DLBCL. This study may be useful to better define specific antigen profile of different PC-BCL entities leading to a correct diagnosis.

New B-cell CD molecules

B cells not only play a pivotal role in humoral immunity, but also are involved in a broad spectrum of immune responses, including antigen presentation and T-cell function regulation. The identification of cell-surface CD molecules derived from a series of Human Leukocyte Differentiation Antigens (HLDA) Workshops has been instrumental to the discovery and functional characterization of human B-cell populations. Moreover, many events regulating B-cell development, activation, and effector functions are orchestrated by these cell-surface molecules. During the Ninth HLDA Workshop (HLDA9) eighteen new CDs were allocated to cell-surface molecules expressed on B cells: CD210a (IL10RA), CD215 (IL15RA), CD270 (TNFRSF14), CD307a (FCRL1), CD307b (FCRL2), CD307c (FCRL3), CD307d (FCRL4), CD351 (FCAMR), CD352 (SLAMF6), CD353 (SLAMF8), CD354 (TREM1), CD355 (CRTAM), CD357 (TNFRSF18), CD358 (TNFRSF21), CD360 (IL21RA), CD361 (EVI2B), CD362 (SDC2), and CD363 (S1PR1). Here we present their expression patterns on leukocytes, including T lymphocytes, NK cells, granulocytes, monocytes, plasmacytoid and monocyte-derived dendritic cells, and several B-cell subsets. These new CD molecules are expressed on B cells at various stages of differentiation; from bone marrow precursor pro-B cells to plasma cells. Three of them, CD307a, CD307b and CD307d, exhibit a B-cell restricted expression pattern, whereas the rest are also present on other leukocytes. In this paper we also review the structural characteristics, expression, and function of these new CD molecules. The availability of monoclonal antibodies directed against novel B cell-surface molecules will have broad implications not only for B-cell biology, but also for the development of new diagnostic and therapeutic tools.