Human Leukocyte Differentiation Antigen Workshop Research Articles

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.

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.

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.

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.

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.

Analysis of HLDA9 mAbs on plasmacytoid dendritic cells

Dendritic cells are a heterogeneous population of bone marrow derived leucocytes that play a crucial role in both pathogen recognition and the initiation of primary T cell immune responses. Plasmacytoid dendritic cells (pDCs), also known as natural interferon-producing cells, comprise one of two major human dendritic cell subsets that strongly influences immune balance. pDCs remain a poorly characterized subset. Several studies have suggested the existence of a close phenotypic and functional relationship between B-cells and pDCs. The surface reactivity of a panel of 96 monoclonal antibodies submitted to the ninth Human Leukocyte Differentiation Antigens workshop (HLDA9) B cell section was analyzed using pDCs as target cells. The results showed that eight of the mAbs reacted positively on pDCs: CD86, CD229, CD319, CD305, CD184, CD84, CD85g and FcɛRIa, confirming previously published reports. Interestingly, this study also revealed the expression of eight surface molecules not previously described on pDCs, including CD352(NTBA), CD272(BTLA), CD357(GITR), CD48, CD270(HVEM), Galectin-3, CD148, and CD361. The present report summarizes the expression of these molecules on freshly isolated pDCs. Significantly, we have identified several new molecules expressed by these intriguing cells, ones we believe will open new avenues for the study of pDC functionality and their role in human health and diseases.

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.

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.

A novel binding assay to assess specificity of monoclonal antibodies

Dynabeads® TALON™ are uniform superparamagnetic polystyrene beads of 1μm diameter that bind 6-His-tagged recombinant proteins through Co ++-affinity binding, and are normally used for protein purification. We have used these beads to bind 6-His-rNKp30 and 6-His-rNKp46 to use as a matrix for evaluating NKp30 and NKp46 mAb submitted to the 8th International Human Leukocyte Differentiation Antigen Workshop. We show that recombinant protein coated beads are an effective tool to evaluate the specificity and epitope reactivity of mAb.

Eighth Leucocyte Differentiation Antigen Workshop DC section summary

Dendritic cells (DC) are specialist antigen presenting cells that play a role in the initiation of innate and adaptive immune response. At the seventh Human Leucocyte Differentiation Antigen workshop, these intriguing cell populations were included as a separate lineage of leucocytes. This paper reports the studies performed in the eighth Human Leucocyte Differentiation Antigen workshop as part of the DC section. Many investigators currently focus on DC that are derived from a number of different leucocyte populations, including those that are differentiated in vitro and cells that are purified ex vivo. The DC section assessed the surface expression of different leucocyte surface molecules on a range of different DC populations. The results summarise the expression of each molecule on dendritic cell populations and differences between different DC preparations. Eleven new CDs were allocated on the basis of monoclonal antibodies and molecular information that identify known cell surface molecules expressed by dendritic cells. This paper gives a brief review of the work that was performed during the HLDA8 and a summary of the CDs represented by submitted mAb.

Characterization of antibodies submitted to the B cell section of the 8th Human Leukocyte Differentiation Antigens Workshop by flow cytometry and immunohistochemistry

The aim of this study was to characterize the reactivity of monoclonal antibodies (mAbs) that had been submitted to the HLDA8 Workshop. The lineage specificity of target molecules was tested by analyzing their expression patterns on blood cells, leukocytes, and lymphocyte subsets. The expression of target molecules during B cell development, ranging from early precursors to plasma cells, was analyzed using a large panel of B cell lines. Our results have permitted us to characterize the expression of 10 new CD molecules: CD316 (HM1.24, BST2), CD268 (BAFF-R, TNFRSF13C), CD269 (BCMA, TNFRF17), CD267 (TACI, TNFRSF13B), CD275 (ICOSL, B7H2), CD254 (TRANCE, TNFSF11), CD252 (OX40L TNFSF4), CD315 (CD9-P), CD316 (EWI-2, PGRL), and CD307 (IRTA-2 or FcRH5). Three of these new CDs, CD267, CD269, and CD307 presented a B cell-restricted expression pattern. MAbs against these novel cell-surface molecules may offer new tools for research, diagnosis, and therapy.

Evaluation of specificity and effects of monoclonal antibodies submitted to the Eighth Human Leucocyte Differentiation Antigen Workshop on rotavirus-cell attachment and entry

Rotavirus infection of permissive cells is a multi-step process that requires interaction with several cell surface receptors. Integrins α2β1, α4β1, αXβ2, and αvβ3 are involved in the attachment and entry into permissive cells for many rotavirus strains. However, possible roles of known partners of these integrins in this process have not been studied. Here, the specificities of new monoclonal antibodies directed to β1 and β2 integrins were determined using integrin-transfected cells. The ability of monoclonal antibodies to integrin partners CD82, CD151, CD321, and CD322 to bind rotavirus-permissive cell lines (MA104, Caco-2, and RD) and K562 cells expressing or lacking α4β1 also was investigated. CD82 and CD151 were expressed on K562, α4-K562, and RD cells. CD321-specific antibodies bound K562, α4-K562, MA104, and Caco-2 cells. CD322 expression was detected on MA104 but not Caco-2 cells. Antibodies to CD82, CD151, CD321, and CD322 that bound these cells were investigated for their ability to inhibit cellular attachment and entry by rotaviruses. Antibody blockade of these integrin-associated proteins did not affect cell attachment or entry of the integrin-using rhesus rotavirus RRV or porcine rotavirus CRW-8, which uses α4β1 integrin for infection. Antibody blockade of CD322 did not alter cell attachment or infectivity by human rotavirus strain RV-3, so RV-3 infection was independent of CD322. Overall, these studies indicate that CD82, CD151, CD321, and CD322 are unlikely to play a role in rotavirus-cell binding or entry.

Reactivity of new adhesion molecules on lymphocytes from patients with chronic graft versus host disease.

Reaction patterns of the 7th Human Leukocyte Differentiation Antigen Workshop blind panel adhesion molecules were studied on CD3/CD4, CD3/CD8, CD3/TCR gamma delta double positive T cells from peripheral blood of patients with chronic graft versus host disease (n = 8) and healthy controls (n = 4). Reactivity of 14 adhesion antibodies was tested by three-colour immunophenotyping. The mean proportion of CD3+ T cells (69 +/- 19%). CD3/CD8++ (31 +/- 13%) and CD3/TCR gamma delta++ (4 +/- 2%) T sub-populations of patients were comparable with the healthy controls. However, the mean percentage of CD3/CD4++ T cell subset in patients (14 +/- 12%) proved to be significantly decreased in comparison with the normal control value (34 +/- 16%) presumably due to secondary immunodeficiency. The workshop antibodies proved to be reactive with three T cell subsets expressing the examined antigens. Based on the results of the adhesion molecule workshop new CD categories have been introduced: CD156b as a transmembrane protein, CD167a as an epithelial tyrosin kinase receptor, CD168 as a receptor for hyaluronan mediated motility (RHAMM) and CD171 as a co-stimulatory adhesion molecule. There were significant differences in the expression of the CD167a and CD156b antigens on the CD3/CD4++ subset between the samples of patients compared with the controls characterizing the CD4+ T lymphocyte subpopulation in chronic graft versus host disease.

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).

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.