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

In the early days of mAb technology, a plethora of human cell surface molecules was identified and described. To avoid confusion and enhance the field, Human Leukocyte Differentiation Antigens (HLDA) Workshops were organized that implemented a standard nomenclature for clusters of Abs that reacted with a specific Ag, providing consistency and uniformity in manuscripts referring to identical molecules. This standardization is commonly referred to as the cluster of differentiation (CD) nomenclature. At present, CD markers range from CD1 to CD371, with some CDs covering a group of closely related proteins or carbohydrates (e.g., CD1a, CD1b, CD1c, and CD1d). In this review, we aim to explain the CD nomenclature system and provide a rationale for its usefulness in an age of rAbs and Ab therapies.

A nondescriptive CD number is assigned to a group or cluster of mAbs that recognize the same cell surface molecule (e.g., CD2 or CD3). The CD designation refers to a group of mAbs shown by the statistical method of cluster analysis to recognize a particular cellular-differentiation pattern. The CD nomenclature is also used to name the molecule itself. For example, CD4 designates both the group of mAbs recognizing the CD4 cell surface molecule, as well as the CD4 molecule itself.

A lowercase “w” preceding the number designation stands for “workshop” (e.g., CDw12) and indicates that the CD designation is tentative; it denotes an insufficiently characterized Ab or molecule. In some cases, it corresponds to a molecule defined by only one Ab submitted to the HLDA Workshops. Most of the provisional CDw-designated Ags of the early workshops turned out to correspond to clusters of mAbs recognizing carbohydrate epitopes, which after proper biochemical identification received their own CD number (e.g., CD176 = Thomsen-Friedenreich, carbohydrate Ag) (1).

Uppercase letters following a CD number designate a spliced variant of the extracellular domain of a cell surface molecule. For example, CD45RA or CD45RO corresponds to splice variants of CD45. A lowercase letter following the CD number (e.g., CD1a, CD1b, CD1c, CD1d, or CD1e) indicates several molecules that share a common chain, in this example, β2-microglobulin. Other examples are the integrin chains CD11a, CD11b, and CD11c, all of which share CD18 as a common chain to form different dimers. In other cases, lowercase letters have been used to name different members of the same gene family, as is the case with CD66 (CD66a, CD66b, CD66c, CD66d, CD66e, and CD66f). With regard to carbohydrate CD structures, a lower case suffix represents a modification of the same carbohydrate sequence (e.g., CD15s = sialylated CD15, Lewis^x Ag; CD60b = 9-O-acetylated ganglioside GD3) (1). Details of carbohydrate CD Ags and carbohydrate-binding CD proteins can be found at http://glycosciences.de/glycocd/index.php.

The CD nomenclature is also frequently used to describe lymphocyte and leukocyte subsets. A “+” symbol is added as a superscript to a CD number to indicate the presence of that molecule on a cell or cell population, and a “−” superscript indicates its absence, as in CD3⁺CD4⁺CD8⁻. If a particular CD molecule is expressed at different levels by a cell subset, the superscript “high” or “low” can be added, as in CD4⁺CD45RA^lowCD45RO^high_.

In the past, an uppercase letter was added to some CDs to group related molecules under the same CD number. This was the case for selectins: CD62L (L-selectin), CD62E (E-selectin), and CD62P (P-selectin). Unfortunately, this turned out to be confusing, because sometimes an “L” was added by some researchers to indicate “ligand,” such as for CD154, commonly referred to as CD40L. To avoid confusion, the addition of uppercase “L” has been discontinued. It should be noted that the terms CD5L, CD20L, and CD137L are not approved nomenclature and should be avoided.

HLDA Workshops were created to establish the nomenclature of leukocyte cell surface molecules by using mAbs from different laboratories. Currently, HLDA Workshops are run by the Human Cell Differentiation Molecules (HCDM) organization (http://www.HCDM.org) under the umbrella of the International Union of Immunological Societies/World Health Organization nomenclature and standardization committees.

With the advent of hybridoma technology to produce mAbs, immunologists began to generate very large numbers of mAbs directed against leukocyte cell surface molecules, generally using whole cells as immunogen. The problem was that several mAbs produced by different laboratories (under different names) were actually directed against the same molecule. This was not always obvious, because the description of the cellular expression pattern reflected the different local interests of the research groups. This resulted in the chaotic naming of molecules, and a Tower of Babel of terminology arose (2). To solve this problem, the first international HLDA Workshop and Conference was organized in 1982 by Alain Bernard and Laurence Boumsell and was cochaired by Jean Dausset, Cesar Milstein, and Stuart F. Schlossman. It was sponsored by INSERM, the Medical Research Council, the World Health Organization, and the International Union of Immunological Societies, mimicking the already existing HLA Workshops that were organized for establishing the nomenclature of HLA alleles (3). The initial goal was to identify groups of mAbs reacting with a common Ag and to agree upon a nomenclature to facilitate better and consistent communication within the scientific community (4). Soon, HLDA workshops proved essential in the identification and characterization of the molecules that populate the surface of hematopoietic cells. The successive workshops provided a forum for the exchange of mAbs and information. Consequently, these workshops were instrumental in unraveling the function of leukocyte cell surface molecules and profoundly transformed our understanding of functional properties of immune cells, as well as their differentiation, maturation, and activation. Ten HLDA Workshops have been organized thus far, with the most recent in 2014 chaired by Georgina Clark.

HLDA Workshops are wet workshops based on an international exchange and blind evaluation of mAbs, submitted by numerous academic laboratories and/or companies. The main goal has consistently been to identify mAbs reacting with a common Ag. The basic strategy was to assess a given mAb’s reactivity with a large panel of different lymphoid cells, followed by statistical analysis of the resulting expression data and further examination of the biochemical nature and molecular mass of the target Ag. Although cellular expression analysis remains essential, modern molecular biology techniques are very useful for a clearer identification of the molecular structures of the target Ags than was possible in the earliest workshops (5).

The initial step consists of establishing one or more panels of mAbs that are submitted by academic groups and/or companies. The organizing laboratory aliquots and distributes the mAbs among the participating laboratories. This has been quite challenging. For example, during HLDA5, >100,000 aliquots of 1,450 mAbs were prepared and distributed among the participating laboratories (6).

Participating laboratories perform specific blind studies with the mAbs included in the panel. This allows for the testing of mAb reactivity with multiple cell types using multiple-color flow cytometry. Because these studies require analysis of huge numbers of different types of primary normal and malignant cells and cell lines, this approach is only possible as a combined effort by a large group of laboratories.

Other participating researchers perform additional tests, such as immunohistochemistry on tissue sections or the biochemical characterization of the target molecules using immunoprecipitation, Western blots, or binding studies to the recombinant target molecules.

The flow cytometry expression data are collected by the organizing laboratory and analyzed using a hierarchical clustering algorithm (3). The biochemical and molecular biological data are used to further validate the clustering analysis.

Currently, the designation of new CDs requires submission to the workshop of at least two independent mAbs that recognize the same molecule and present an identical pattern of reactivity. Proof of specific reactivity with transfected cells is mandatory to obtain a CD designation. Such mAbs must specifically recognize the target protein on transfected cells, as well as the endogenous protein on live primary cells. During the last two HLDA Workshops, the cross-reactivity of the Abs with proteins encoded by a common gene family also had to be tested. This is essential if the degree of homology between molecules of the same family is very high. Thus, it is mandatory to exclude cross-reactivity with other related Ags or proteins encoded by other members of the same gene family.

Some participating laboratories test the agonistic or antagonistic effect of the Abs in a variety of functional assays, such as proliferation, apoptosis, or adhesion blocking. Thus, the HLDA Workshop protocol allows the assignment of CD names, and it generates an enormous amount of data on the reactivity of the Abs and the expression and functional characteristics of the target molecules. It represents a very efficient and comprehensive way to independently validate a mAb.

All of the generated data and experiments performed with the submitted mAbs are presented at the HLDA Conferences. These conferences are a valuable forum for discussion of the function of cell surface molecules. A list of the proceedings and references of the different HLDA Conferences and a database with mAbs that were approved by the HLDA Workshops are available at http://www.HCDM.org.

Table I shows the current official list of CD molecules. The total number of assigned CDs is 401. The most recent CD designations to be assigned (CD365 to CD371) were established at the HLDA10 Workshop (held in Wollongong, Australia in December 2014). Some CD numbers were dropped because their molecular nature could not be confirmed, or their designation was reassigned (e.g., CDw12, CD67, and CD78). A second set of missing CDs corresponds to CD numbers reserved for molecules for which no useful mAbs have been validated by the HLDA Workshops. These include CD285, CD287, and CD291, corresponding to TLRs, as well as CD255, reserved for TNFF12. The CD label is only valid for those mAbs that have been scrutinized in HLDA Workshops and have fulfilled high-quality criteria. Unfortunately, there are a number of mAbs on the market that misuse the CD label, creating confusion in data interpretation.

Directly from the start, the HLDA Workshops were very exciting for immunologists, because they provided important discovery tools that deliver an element of confidence in the world of reagents. The impact of the workshops has been enormous, both in basic immunology and in clinical practice, as exemplified by the applications in clinical diagnosis and treatment of malignancies and immunological diseases (7, 8). Another relevant outcome has been the synergy between research groups and industry. Many companies have commercialized mAbs that researchers have submitted to the HLDA Workshops. More recently, companies have become an important source of mAbs for HLDA Workshops.

Bioinformatics studies indicate that we have characterized only a fraction, probably just one third, of the cell surface molecules expressed by the cells of the immune system (9, 10). This may be due, in part, to the fact that most of the CDs were defined with mAbs generated from rodents. Direct protein profiling, which reflects the actual levels of protein expression, is limited by the availability of high-quality mAbs against these molecules. Thus, the main goal of the HLDA Workshops is to continue with the characterization of all of the cell surface molecules relevant to the immune system and to immune-mediated diseases and the validation of mAbs against these molecules.

Moreover, there are extensive gaps in our knowledge of CD molecule expression patterns, mainly because of the heterogeneity of the expression studies and the significant changes in flow cytometry technology over the last 30 y. HCDM recently launched a new project called CDMaps, whose objective is to systematically and accurately determine the expression patterns of established CD molecules on all major blood and lymphoid tissue leukocyte subsets, using state-of-the-art multicolor flow cytometry. The use of standardized instrument settings and immunophenotyping protocols with HLDA-approved reagents will generate a data set that is highly reproducible among laboratories (11). Several companies are supporting this project by providing mAbs and the backbone testing cocktails for these tests (http://www.hcdm.org/images/PDF/CDMaps.pdf). Through additional quantification of the number of molecules/cell type, the CDMaps project should generate an enormous database of protein expression profiles that will represent a unique open access resource for basic, translational, and clinical immunology.

Finally, there is an urgent need for independent validation of mAbs. The production of mAbs has become a routine procedure. In recent years, an overwhelming number of mAbs against leukocyte cell surface molecules, as well as other target molecules, have been produced by academic groups and an increasing number of companies. However, a large number of these Abs remain poorly validated. One common observation is that mAbs often recognize recombinant proteins, typically the immunogen, but are unable to react with the endogenous or native protein on live primary cells. The use of improperly validated Abs has resulted in the loss of thousands of work hours, destroyed research projects, and generated false results that have contaminated the scientific literature. The scientific community is increasingly aware of this extremely serious problem (12). Toward the aim of solving this problem, the HLDA Workshop protocol guarantees the validation of submitted mAbs and, therefore, represents an invaluable tool for safeguarding our knowledge of CD molecules and their role in the immune system. This is the reason why the U.S. Food and Drug Administration requested that, for a mAb to be used as a diagnostic reagent, it should be evaluated by the HLDA Workshops. We propose in this article that HLDA Workshop evaluation should be made mandatory for therapeutic mAbs, particularly for biosimilar mAbs targeting leukocyte cell surface molecules. In addition, we suggest adding the CD nomenclature to the International Nonproprietary Name used to designate original and biosimilar mAbs.

HLDA Workshops are a great example of an effective long-term international collaboration across scientific disciplines and laboratories. Future workshops will continue to promote the exchange of reagents between academic groups and industry and will aim to boost the supply of highly characterized mAbs for all cell surface molecules. These workshops will provide a platform to increase our understanding of leukocyte biology and pathology, as well as facilitate the identification of new disease biomarkers and therapeutic targets.

R.B. is an employee of BD Biosciences and F. Mortari of Bio-Techne (R&D Systems). The rest of the authors report no conflict of interest.

We thank the International Union of Immunological Societies for its continuous support of the CD Molecule Nomenclature and Leukocyte Standardization Subcommittees.