CD molecules 2005: human cell differentiation molecules

The Human Leukocyte Differentiation Antigens (HLDA) Workshops were initiated¹  to bring order to the chaos that existed in the early 1980s, as immunologists generated large numbers of monoclonal antibodies reactive with leukocyte cell-surface molecules, each with different associated nomenclatures. In the absence of comparative studies it was often impossible to tell if the same molecule was recognized by more than one antibody. The approach of the workshops was to code antibodies and then send them to multiple participating laboratories for blind analysis against multiple cell types. The data were collated and analyzed by the statistical procedure of “cluster analysis.” This analytical method identified clusters of antibodies with very similar patterns of binding to leukocytes at various stages of differentiation: hence the “cluster of differentiation” (CD) nomenclature. This provided a common nomenclature that allowed the scientific community to communicate results in a universal language. The full list of CD molecules may be accessed through the 8th HLDA Workshop (HLDA8) website (www.hlda8.org), which will be replaced by a comprehensive web-based database providing links to protein and gene web-based databases (I.N. and V.H., in preparation).

In the early workshops, the raw material comprised the large numbers of submitted antibodies against unknown molecules. Statistical cluster analysis, together with protein biochemical techniques such as immunoprecipitation, then provided the major tools for identifying the new molecules defined by workshop antibodies. Many of the molecules used every day by immunologists, hematologists, and pathologists were discovered this way, including CD3, CD4, CD8, and CD20. In addition, the workshops provided a forum for the study of function and distribution of molecules and epitopes, and catalyzed the development of an industry providing reagents for research, diagnosis, and now therapy.² 

However, as molecular biologic techniques grew in power, new molecules present on leukocytes were increasingly identified via gene cloning. Antibodies began to be made after the molecule had been cloned, rather than before. Monoclonal antibodies remain useful for research and diagnosis—and are of increasing importance for therapy—but they no longer represent the primary tool for discovery of new proteins (in contrast to glycolipid and carbohydrate epitopes, where antibodies remain the major tool for identifying new molecules).

While these changes progressed, HLDA workshops have continued to be held in a 4-year cycle, with the major aim of allocating CD numbers to well-characterized molecules against which good-quality antibodies are available. However, there has been increasing debate, within as well as outside the HLDA organization, on how appropriate this role may be in the era of molecular proteomics. These discussions culminated in agreement at the meeting of the HLDA Council in December 2004 to make radical changes, including a change in name as well as aims and methods. These are summarized at the end of this article.

HLDA8 adopted the aims and methodology established at previous workshops (since the changes detailed at the end of this article were not agreed on until after the meeting in December 2004). The major aims were (1) to assign CD names to molecules for which good-quality antibodies (at least one) and good molecular data are available (generally meaning that the molecule has been cloned); (2) to validate new antibodies against existing CD molecules; (3) to evaluate a “blind panel” of new monoclonal antibodies with a view to identifying new molecules by cluster analysis, following the traditional approach described above; and (4) to provide a forum for discussing and developing scientific understanding and practical applications of leukocyte differentiation molecules, including their ligands.

Table 1 lists the 95 molecules that were assigned CD numbers as a consequence of the HLDA8 meeting. The antibodies validated against these “new” CD molecules, as well as newly validated antibodies against preexisting CD molecules, are listed on the HLDA8 website (www.hlda8.org).

A blind antibody panel was evaluated along the lines of previous HLDA blind panels.^3-6  Although a number of pairs and groups of antibodies were found to have very similar reactivity with the panel of cells, we were not able to assign any new CD molecules on the basis of these studies. This was due in part to incomplete biochemical studies, and these are continuing after the completion of the HLDA8 meeting. However, the experience of the nonlineage and adhesion/stromal cell sections, in which a number of antibodies against apparently new molecules turned out to be against existing CD markers, served to emphasise the ever-increasing risk of “rediscovering” known molecules. As a consequence, the HLDA Council decided that, in the future, new CDs deriving from blind-panel analysis would require definitive molecular identification of the target antigen. This may be performed by using the antibody to “expression clone” the corresponding gene or to immunoprecipitate the antigen for mass spectrometric analysis.

The final aim, to provide a forum for discussion and development of the field, was accomplished in part by the 8th HLDA Conference that accompanied the workshop and was held jointly with the Australasian Society for Immunology. This aim will also be achieved by this report and by a number of publications that will follow the workshop. In keeping with current practice, much of the information of archival value (in particular validated antibodies) will be available through the web (www.hlda8.org).

The newly assigned CD numbers are shown in Table 1.

The question of whether HLDA has reached the point of diminishing returns is raised after every workshop. However, there is good reason to believe we have so far accounted for just a fraction of the molecules expressed on leukocyte surfaces,⁷  and the 95 new designations of HLDA8 support that view. Although there is plenty still to do, are we still going about it in the right way? In recognition of changes in biology generally and in the field served by HLDA, the HLDA Council reached several decisions.

The acronym “HLDA” will be succeeded by “HCDM” (for “human cell differentiation molecules”). The reasoning behind this name change is as follows. (1) To indicate a break with tradition, while retaining the letters “CD.” (2) To maintain the emphasis on molecules of human origin (although the HCDM project will continue to provide leadership and a forum for collaborative studies in other species). (3) To extend the focus from leukocytes to other cell types, recognizing that leukocytes do not act alone. The HLDA project has fostered studies on endothelial molecules for some years, since they interact with cells of the immune system. Furthermore, the HLDA8 meeting included an active section devoted to studies of stromal cell molecules. We therefore believe that the multidisciplinary approach pioneered by previous HLDA workshops can be applied successfully to many other nonhematopoietic human cell types. (4) To broaden the scope from cell-surface molecules to any molecule whose expression reflects differentiation. This would recognize the growing value of intracellular molecules, particularly in immunohistologic studies of tissue sections, as markers of differentiation.

The HLDA Council is a subcommittee of the International Union of Immunological Societies (IUIS) Nomenclature Committee, charged with responsibility for generating a consensus nomenclature for leukocyte cell-surface molecules. The HCDM Council will retain this role (subject to the wishes of IUIS) and HCDM will give appropriate consideration to gene nomenclature assigned by the Human Genome Organization (HUGO) Gene Nomenclature Committee. CD numbers will not be allocated as routine practice to all leukocyte surface molecules recognized by newly available antibodies. CD numbers will continue to be allocated to molecules where this appears likely to facilitate communication.

Validation of antibodies against known molecules has always been a central aspect of the HLDA organization. This remains an important function: users of commercial antibodies would like the reagents they purchase to be validated by an independent laboratory, since, in spite of technical advances, antibodies are still frequently misassigned by their creators. The HCDM project will therefore undertake this validation role, and do so on a systematic basis. Furthermore, it will not restrict itself to molecules that subsequently receive a CD designation; that is, the nexus between antibody validation and nomenclature will be broken. The HCDM office will maintain a database of all validated hybridoma clones, and will also seek to ensure that validated antibodies are available in practice to the research community, whether commercially or otherwise. However, there is a practical limit to the role that HCDM can play in antibody validation. Antibodies can be checked for specificity, and only antibodies that give a clear-cut positive result will be recognized as workshop-validated. However, fitness for particular uses and the provision of antibody at suitable concentrations and in suitable forms (including, for example, conjugates) remains a matter for the companies marketing antibodies.

The HCDM laboratories will validate antibodies in a yearly cycle, and the validation of new antibodies and any CD designations accepted by the Council will be posted on the HCDM website at the end of each year. A web-based set of databases will be maintained, including a listing of all antibodies that have been validated through the workshop process, and a database providing basic information for each molecule, together with links to other web-based information resources, allowing quick access to molecular and distribution data.

The HLDA initiative has served immunology, hematology, and pathology well over the 22 years that have elapsed since the first workshop, and it provided much-needed clarity in the chaotic early days of monoclonal antibodies. The order wrought by the pioneers of HLDA studies has supported innumerable research, diagnosis, and therapy initiatives by providing independent confirmation of antibody specificity, as well as a universal nomenclature scheme. However, advances in technology and knowledge have meant that the HLDA initiative needs to change to maintain its relevance. The recent HLDA8 meeting created 95 new CD assignments, but it also defined radical changes in the aims and methods of the organization. This has positioned the new HCDM organization to play a vital role in future exciting antibody-based studies that will discover and exploit many new functional human proteins.

We thank the State Cancer Council of South Australia, The Australian Government, International Science Linkages Programme, Becton Dickinson, DakoCytomation, R&D Systems, Beckman Coulter, Serotec, Miltenyi Biotec, Diaclone, and Caltag for their support throughout the project.