Abstract

The immune system works through leukocytes interacting with each other, with other cells, with tissue matrices, with infectious agents, and with other antigens. These interactions are mediated by cell-surface glycoproteins and glycolipids. Antibodies against these leukocyte molecules have provided powerful tools for analysis of their structure, function, and distribution. Antibodies have been used widely in hematology, immunology, and pathology, and in research, diagnosis, and therapy. The associated CD nomenclature is commonly used when referring to leukocyte surface molecules and antibodies against them. It provides an essential classification for diagnostic and therapeutic purposes. The most recent (8th) Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA), held in Adelaide, Australia, in December 2004, allocated 95 new CD designations and made radical changes to its aims and future operational strategy in order to maintain its relevance to modern human biology and clinical practice.

Introduction

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.

Materials and methods

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).

Table 1.

List of new CD assignments


New CD
 

Molecule
 

Gene ID*
 

Gene symbol
 
CDw113   PVRL3, Nectin3   25945  PVRL3 
CD118   LIFR   3977  LIFR 
CDw156C   ADAM10   102  ADAM10 
CD159c   NKG2C   3822  KLRC2 
CD172b   SIRPbeta   10326  SIRPB1 
CD172g   SIRPgamma   55423  SIRPB2 
CD181 (was CDw128A)   CXCR1   3577  IL8RA 
CD182 (was CDw128B)   CXCR2   3579  IL8RB 
CD185   CXCR5   643  BLR1 
CDw186   CXCR6   10663  CXCR6 
CD191   CCR1   1230  CCR1 
CD192   CCR2   1231  CCR2 
CD193   CCR3   1232  CCR3 
CD196   CCR6   1235  CCR6 
CD197   CCR7   1236  CCR7 
CDw198   CCR8   1237  CCR8 
CDw199   CCR9   10803  CCR9 
CDw218a   IL18Ralpha   8809  IL18R1 
CDw218b   IL18Rbeta   8807  IL18RAP 
CD248   TEM1, Endosialin   57124  CD164L1 
CD249   Aminopeptidase A   2028  ENPEP 
CD252   OX40L   7292  TNFSF4 
CD253   TRAIL   8743  TNFSF10 
CD254   TRANCE   8600  TNFSF11 
CD256   APRIL   8741  TNFSF13 
CD257   BLYS   10673  TNFSF13B 
CD258   LIGHT   8740  TNFSF14 
CD261   TRAIL-R1   8797  TNFRSF10A 
CD262   TRAIL-R2   8795  TNFRSF10B 
CD263   TRAIL-R3   8794  TNFRSF10C 
CD264   TRAIL-R4   8793  TNFRSF10D 
CD265   TRANCE-R   8792  TNFRSF11A 
CD266   TWEAK-R   51330  TNFRSF12A 
CD267   TACI   23495  TNFRSF13B 
CD268   BAFFR   115650  TNFRSF13C 
CD269   BCMA   608  TNFRSF17 
CD271   NGFR (p75)   4804  NGFR 
CD272   BTLA   151888  BTLA 
CD273   B7DC, PDL2   80380  PDCD1LG2 
CD274   B7H1, PDL1   29126  PDCD1LG1 
CD275   B7H2, ICOSL   23308  ICOSL 
CD276   B7H3   80381   NA  
CD277   BT3.1   11119  BTN3A1 
CD278   ICOS   29851  ICOS 
CD279   PD1   5133  PDCD1 
CD280   ENDO180   9902  MRC2 
CD281   TLR1   7096  TLR1 
CD282   TLR2   7097  TLR2 
CD283   TLR3   7098  TLR3 
CD284   TLR4   7099  TLR4 
CD289   TLR9   54106  TLR9 
CD292   BMPR1A   657  BMPR1A 
CDw293   BMPR1B   658  BMPR1B 
CD294   CRTH2   11251  GPR44 
CD295   LeptinR   3953  LEPR 
CD296   ART1   417  ART1 
CD297   ART4   420  DO 
CD298   Na+/K+-ATPase β3 subunit   483  ATP1B3 
CD299   DCSIGN-related   10332  CD209L 
CD300a   CMRF35H   11314   NA  
CD300c   CMRF35A   10871   NA  
CD300e   CMRF35L1   N/A   NA  
CD301   MGL, CLECSF14   10462  CLECSF14 
CD302   DCL1   9936   NA  
CD303   BDCA2   170482  CLECSF7 
CD304   BDCA4, Neuropilin 1   8829  NRP1 
CD305   LAIR1   3903  LAIR1 
CD306   LAIR2   3904  LAIR2 
CD307   IRTA2   83416   NA  
CD309   VEGFR2, KDR   3791  KDR 
CD312   EMR2   30817  EMR2 
CD314   NKG2D   22914  KLRK1 
CD315   CD9P1   5738  PTGFRN 
CD316   EWI2   93185  IGSF8 
CD317   BST2   684  BST2 
CD318   CDCP1   64866   NA  
CD319   CRACC   57823  SLAMF7 
CD320   8D6A   51293   NA  
CD321   JAM1   50848  F11R 
CD322   JAM2   58494  JAM2 
CD324   E-Cadherin   999  CDH1 
CDw325   N-Cadherin   1000  CDH2 
CD326   Ep-CAM   4072  TACSTD1 
CDw327   siglec6   946  SIGLEC6 
CDw328   siglec7   27036  SIGLEC7 
CDw329   siglec9   27180  SIGLEC9 
CD331   FGFR1   2260  FGFR1 
CD332   FGFR2   2263  FGFR2 
CD333   FGFR3   2261  FGFR3 
CD334   FGFR4   2264  FGFR4 
CD335   NKp46   9437  NCR1 
CD336   NKp44   9436  NCR2 
CD337   NKp30   259197  NCR3 
CDw338   ABCG2, BCRP   9429  ABCG2 
CD339
 
Jagged-1
 
182
 
JAG1
 

New CD
 

Molecule
 

Gene ID*
 

Gene symbol
 
CDw113   PVRL3, Nectin3   25945  PVRL3 
CD118   LIFR   3977  LIFR 
CDw156C   ADAM10   102  ADAM10 
CD159c   NKG2C   3822  KLRC2 
CD172b   SIRPbeta   10326  SIRPB1 
CD172g   SIRPgamma   55423  SIRPB2 
CD181 (was CDw128A)   CXCR1   3577  IL8RA 
CD182 (was CDw128B)   CXCR2   3579  IL8RB 
CD185   CXCR5   643  BLR1 
CDw186   CXCR6   10663  CXCR6 
CD191   CCR1   1230  CCR1 
CD192   CCR2   1231  CCR2 
CD193   CCR3   1232  CCR3 
CD196   CCR6   1235  CCR6 
CD197   CCR7   1236  CCR7 
CDw198   CCR8   1237  CCR8 
CDw199   CCR9   10803  CCR9 
CDw218a   IL18Ralpha   8809  IL18R1 
CDw218b   IL18Rbeta   8807  IL18RAP 
CD248   TEM1, Endosialin   57124  CD164L1 
CD249   Aminopeptidase A   2028  ENPEP 
CD252   OX40L   7292  TNFSF4 
CD253   TRAIL   8743  TNFSF10 
CD254   TRANCE   8600  TNFSF11 
CD256   APRIL   8741  TNFSF13 
CD257   BLYS   10673  TNFSF13B 
CD258   LIGHT   8740  TNFSF14 
CD261   TRAIL-R1   8797  TNFRSF10A 
CD262   TRAIL-R2   8795  TNFRSF10B 
CD263   TRAIL-R3   8794  TNFRSF10C 
CD264   TRAIL-R4   8793  TNFRSF10D 
CD265   TRANCE-R   8792  TNFRSF11A 
CD266   TWEAK-R   51330  TNFRSF12A 
CD267   TACI   23495  TNFRSF13B 
CD268   BAFFR   115650  TNFRSF13C 
CD269   BCMA   608  TNFRSF17 
CD271   NGFR (p75)   4804  NGFR 
CD272   BTLA   151888  BTLA 
CD273   B7DC, PDL2   80380  PDCD1LG2 
CD274   B7H1, PDL1   29126  PDCD1LG1 
CD275   B7H2, ICOSL   23308  ICOSL 
CD276   B7H3   80381   NA  
CD277   BT3.1   11119  BTN3A1 
CD278   ICOS   29851  ICOS 
CD279   PD1   5133  PDCD1 
CD280   ENDO180   9902  MRC2 
CD281   TLR1   7096  TLR1 
CD282   TLR2   7097  TLR2 
CD283   TLR3   7098  TLR3 
CD284   TLR4   7099  TLR4 
CD289   TLR9   54106  TLR9 
CD292   BMPR1A   657  BMPR1A 
CDw293   BMPR1B   658  BMPR1B 
CD294   CRTH2   11251  GPR44 
CD295   LeptinR   3953  LEPR 
CD296   ART1   417  ART1 
CD297   ART4   420  DO 
CD298   Na+/K+-ATPase β3 subunit   483  ATP1B3 
CD299   DCSIGN-related   10332  CD209L 
CD300a   CMRF35H   11314   NA  
CD300c   CMRF35A   10871   NA  
CD300e   CMRF35L1   N/A   NA  
CD301   MGL, CLECSF14   10462  CLECSF14 
CD302   DCL1   9936   NA  
CD303   BDCA2   170482  CLECSF7 
CD304   BDCA4, Neuropilin 1   8829  NRP1 
CD305   LAIR1   3903  LAIR1 
CD306   LAIR2   3904  LAIR2 
CD307   IRTA2   83416   NA  
CD309   VEGFR2, KDR   3791  KDR 
CD312   EMR2   30817  EMR2 
CD314   NKG2D   22914  KLRK1 
CD315   CD9P1   5738  PTGFRN 
CD316   EWI2   93185  IGSF8 
CD317   BST2   684  BST2 
CD318   CDCP1   64866   NA  
CD319   CRACC   57823  SLAMF7 
CD320   8D6A   51293   NA  
CD321   JAM1   50848  F11R 
CD322   JAM2   58494  JAM2 
CD324   E-Cadherin   999  CDH1 
CDw325   N-Cadherin   1000  CDH2 
CD326   Ep-CAM   4072  TACSTD1 
CDw327   siglec6   946  SIGLEC6 
CDw328   siglec7   27036  SIGLEC7 
CDw329   siglec9   27180  SIGLEC9 
CD331   FGFR1   2260  FGFR1 
CD332   FGFR2   2263  FGFR2 
CD333   FGFR3   2261  FGFR3 
CD334   FGFR4   2264  FGFR4 
CD335   NKp46   9437  NCR1 
CD336   NKp44   9436  NCR2 
CD337   NKp30   259197  NCR3 
CDw338   ABCG2, BCRP   9429  ABCG2 
CD339
 
Jagged-1
 
182
 
JAG1
 

NA indicates not available.

*

Gene ID refers to the Entrez gene accession number.8 

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).

Results

The newly assigned CD numbers are shown in Table 1.

Discussion

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.

Name change

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.

Nomenclature role

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.

Antibody validation role

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.

Dissemination of information

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.

Conclusion

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.

Prepublished online as Blood First Edition Paper, July 14, 2005; DOI 10.1182/blood-2005-03-1338.

H.Z. designed the study and wrote the text; B.S. and D.M. designed the study, analyzed data, and wrote the text; I.N., L.B., P.E., D.H., V.H., and F.M. designed the study and analyzed data; B.A., A.B., C.B., G.C., C.I., P.M., D.O., A.S., M.U., and H.W. designed the study, performed research, and analyzed data; K.D. performed research; S.F.S., R.S.-A., P.S., and T.F.T. designed the study.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

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.

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