The Tradition of Human Leukocyte Differentiation Antigen Workshops

The process of categorizing the antigenic molecules and epitopes associated with human white cells, via the collaborative study of monoclonal antibodies, dates back to the early 1980s, when the first HLDA (Human Leukocyte Differentiation Antigen) Workshop was held in Paris. This initial meeting listed only 15 agreed molecular entities, but it created an internationally agreed basis for the nomenclature of leukocyte molecules (the CD scheme) and also provided a forum for reporting studies on their function and practical relevance. A further six HLDA meetings have been held since the first Paris meeting. The most recent of these (HLDA7) took place last year in Harrogate, UK, and the proceedings of the meeting will be published later this year (Leucocyte Typing VII, Oxford University Press).

The Aims and Approaches of the 7th HLDA Workshop

The Limitations of “Blind” Antibody Screening

It was apparent at the previous meeting, HLDA6, held in Kobe, Japan, in 1996, that the technique of detecting molecular entities by screening coded panels of monoclonal antibodies against human cells was becoming obsolescent. Antibodies to the most immunogenic molecules had already been produced, and fewer laboratories than in the early days were prepared to devote resources to raising new antibodies because the probability of finding novel reagents becomes ever less likely. In consequence, many antibodies in the 6th Workshop were reagents (submitted by laboratories that were not equipped to characterize them) that proved to be of known specificity.

Selection of Antibodies

With these considerations in mind, the 7th Workshop adopted a different approach: instead of screening poorly characterized antibodies, reagents were selected (and actively solicited) for which at least some molecular data were already available. A substantial number of monoclonal antibodies reactive with leukocyte-associated molecules exist that do not meet the traditional criterion for establishing a new CD specificity (i.e., the existence of at least two independent antibodies of the same specificity). This rule dates from the first HLDA Workshop two decades ago: since that time, biochemical and molecular biological techniques for characterizing the targets of new antibodies have come to be widely used. Consequently, it is now considered appropriate to establish a CD designation for a molecule if its gene has been cloned and at least one specific monoclonal antibody has been studied in the Workshop.

New Workshop Sections

Four new sections were introduced in the 7th HLDA Workshop to add to the traditional list from past meetings: namely, Dendritic Cells, Stem/Progenitor Cells, Erythroid Cells, and Carbohydrate Structures. Although it has been recognized for many years that monoclonal antibodies reactive with human leukocytes can be specific for carbohydrate epitopes (e.g., the carbohydrate CD category CD15 was identified at the first Workshop), they had not received specific attention in any Workshop. The inclusion of erythroid molecules, although it may seem out of place in a “Leukocyte Workshop,” was justified by the number of molecules shared between white and red cells (e.g., cytokine receptors) that hint at unexplored functions of red cells.

The Yield of New CD Specificities in the 7th HLDA Workshop

This more active approach to the identification of new CD specificities represented a break with tradition, but the results justified the new approach because a total of well over 80 new entities were added to the list of CD specificities. This compares favorably with previous Workshops (an average of less than 30 CD specificities per Workshop), and it also largely avoided the laborious screening in multiple laboratories of antibodies that prove to be directed against known CD molecules.

Tables 1 and 2 list the new specificities established at the 7th Workshop. Full details will be found in Leucocyte Typing VII, and molecular, functional, and other data can be found for many of these new specificities on the Website for “Protein Reviews on the Web” (www.ncbi.nlm.nih.gov/prow/).

TABLE 1 New CD Designations
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TABLE 2 New CD Nomenclature for ILT/LIR and KIR Molecules
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The Eighth Workshop

Plans are well advanced for the 8th Workshop, to be organized in Adelaide in 2004 under the aegis of Prof. H. Zola (see www.hlda8.org). It is sometimes assumed that the catalog of surface molecules associated with human hemopoietic cells is now essentially complete, but there is abundant evidence in the literature for novel surface molecules that would merit study at the next Workshop and that could provide the basis for new CD designations. Table 3 comprises a list of potential new molecules reported after the production of monoclonal antibodies and also a more extensive list of surface molecules identified by gene cloning. In most instances, no antibodies are available against the putative new leukocyte/endothelial markers in this latter group. Specific and well-characterized reagents, whether monoclonal or polyclonal, are needed not only for detecting these new “virtual” molecules but also for defining functional domains, for characterizing 3D protein structure, and for analyzing protein–protein interactions. It may be added that cloning of gene sequences often reveals multiple members of new or existing molecular families (e.g., the Toll-like receptors) and may identify surface receptors that bind more than one ligand or vice versa (e.g., the TALL-1 and APRIL ligands for TACI and BCMA). Furthermore, a number of leukocyte-associated markers have been cloned from mice and other species, and almost all will have human homologues. The 8th Workshop will provide a forum for a range of antibody-based studies relating to this accumulating corpus of genomic and proteomic data.

TABLE 3 Examples of Possible Future CD Specificities
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As in the 7th Workshop, in which four new sections were added, it may be possible to include neuronal cells in the 8th Workshop. Many neuronal cells express cell surface proteins found on leukocytes and vice versa (e.g., CD56, CD100, CD168, CD171). Furthermore, the guidance cues used by neuronal cells share similarities to those involved in leukocyte extravasation, so the expression of these molecules in common may reflect shared biological processes. It may also be noted that other molecules such as the mucins, thought to be primarily associated with epithelial cells, are now being described on leukocytes.

Finally, it remains to be established how the 8th and subsequent HLDA Workshops should deal with lineage- or stage-restricted leukocyte molecules that are localized within the cell cytoplasm (or nucleus). Given the importance of many of these molecules in signaling pathways initiated via known surface CD molecules, their identification and study is an inevitable extension of the work of the first seven HLDA Workshops. Whether or not a new “intracellular CD” categorization scheme is devised for such molecules, they are of interest for many laboratories studying human hematopoietic cells, and their investigation will be among the aims of the next Workshop.