1 Leukocyte Membrane Molecules—An Introduction 1.1 HISTORY “Workshop conditions” (multiple labo- ratories examining coded panels of anti- In the late 1970s and early 1980s, as immu- bodies), demonstrated that the antibodies nologists came to appreciate the power of all reacted with the same antigen. The monoclonal antibodies as reagents, large differences in the individual descriptions numbers of new antibodies were made, reflected differences in technique, antibody analyzed, and published. It was difficult to affinity, and interpretation. know whether two different antibodies A small group of immunologists recog- were directed against the same antigen.The nized the problem and devised a solution: antigen that was later named CD9 is a good multi-laboratory blind analysis and statisti- example. Five antibodies were described cal evaluation of the results. This solution independently; they shared some features: led to the First International Workshop on precipitation of a protein band of 24–26kD, Human Leukocyte Differentiation Anti- reactivity with platelets, and non-T-non-B gens (HLDA), organized in Paris by Lau- acute lymphoblastic leukemia. There were rence Boumsell and Alain Bernard in 1984 some apparently important differences: (Bernard et al., 1984). The purpose of this one antibody was described as reacting and subsequent workshops was to stan- with 26% of normal blood lymphocytes, dardize reagents and, through the use of whereas the other four were reported to standardized reagents, to develop an under- react with less than 2%; only one of the five standing of the structure and function of was reported to react with monocytes and leukocyte cell surface molecules and their polymorphs; there were differences in utility as diagnostic and therapeutic targets. reported reactivity with chronic lympho- Important tools in this process were a cytic leukemia. Later analysis, under nomenclature (the CD nomenclature) and Leukocyte and Stromal Cell Molecules: The CD Markers, by Heddy Zola, Bernadette Swart, Ian Nicholson, and Elena Voss Copyright © 2007 by John Wiley & Sons, Inc. 3 4 LEUKOCYTE MEMBRANE MOLECULES—AN INTRODUCTION the publication of books (Leukocyte antigen. However, antibody was still the Typing I-VII), which contained the conclu- primary tool for antigen discovery and sions of the workshops and the data on characterization. which these conclusions were based. As molecular technologies improved, new human proteins were increasingly identified by first cloning the gene, either 1.1.1 The Significance of the CD through sequence homology with a gene of Nomenclature and HLDA Workshops animal origin or by searching for genes The CD nomenclature, supported by the with sequence similarity to known mole- HLDA workshops and the Leukocyte cules. With increasing frequency, the anti- Typing volumes, has become accepted body was made after the protein had been universally. All immunology journals and identified, expressed, and characterized. journals in other fields that deal with these The quality and reliability of good mol- molecules use the CD nomenclature. The ecular data has changed the methodologic U.S. Food and Drug Administration (FDA) focus of the HLDA workshops. The has mandated the workshops in the sense primary focus of HLDA has moved to the that antibodies that seek approval as diag- functional molecules (the “antigens”); nostic reagents against CD molecules must the antibodies are tools used in their study. have been validated through the HLDA In the late 1970s and early 1980s, the ques- workshops. The World Health Organiza- tion behind many studies was “What does tion (WHO) and the International Union my antibody react with?” Today we can of Immunological Societies have mandated focus on much more fundamental ques- the CD nomenclature. tions about intercellular interactions and Nevertheless, there have been major cell–molecule interactions. The ligands and changes in the nature and importance of receptors involved in these interactions still the contribution made by HLDA work- need to be described, detected, and mea- shops in the years since the first one was sured, and monoclonal antibodies are still conducted. The main business of the early the most powerful tools for achieving an HLDA workshops was to identify new understanding of these interactions. human leukocyte cell surface molecules, using antibodies that had been made by 1.1.2 Why Does HLDA Focus on immunizing mice with whole cells or cell Surface Membrane Molecules? membranes. The major tools were analysis of reactivity of antibodies with a variety of The early studies (1970s–1980s) focused on cell types and statistical analysis of the the cell surface simply because that was the resulting expression data. The statistical major focus of immunology at the time— procedure used to identify antibodies with immunologists wanted to know how cells a high probability of reacting with the same interacted with other lymphocytes, with molecule was cluster analysis, hence, the endothelium, and with antigen.Without the name “Cluster of Differentiation.” Infor- tools to identify, visualize in tissue, and mation of a confirmatory nature was often isolate individual cell types, many of the contributed by protein analysis—Western questions that have engaged immunolo- blotting or immunoprecipitation followed gists for the last generation could not even by gel electrophoresis. By the third HLDA be formulated, let alone addressed. Diag- workshop, antibodies were also being used nostic distinctions, for example, between to identify antigen expressed in cDNA different types of leukemia, seemed at the libraries, allowing the cloning of the cDNA time to be capable of being addressed with and molecular characterization of the cell surface markers. From the start of the HISTORY 5 monoclonal antibody era (and indeed We cannot be sure, but the evidence sug- earlier, using polyclonal antisera), therapy gests that we are less than halfway there. using antibodies to knock out cancer cells Estimates of the numbers of surface mole- or immune cells (to prevent organ graft cules expressed by one functional category rejection) was a major driver of research, of leukocyte, the T lymphocytes, arrive at and here the targets were clearly cell a number around 1000 (Zola and Swart, surface molecules. 2003). The number of T cell markers cur- Once we had an extensive catalog of cell rently known to be on T cell surfaces is surface molecules, and the tools with which 100–200. The T cells are the best character- to study their function, cell signaling ized leucocytes, so the journey is unlikely to became a new front in the development of be more advanced in the other leukocyte understanding of the immune system. The families. myriad intracellular molecules—syk, zap, The estimate of the total number of mol- cbl, and their ilk—cry out for a nomencla- ecules is based on counting of proteins or ture accessible to scientists from other messenger RNA species expressed by cells fields. The HLDA considered on several and multiplying by the estimated propor- occasions whether to extend the CD system tion of expressed proteins that are mem- to cover these molecules and decided on brane proteins. Some known proteins that several occasions not to. The reasons have not been identified on T cells may included an anxiety that if the CD nomen- nevertheless be expressed on T cells. In clature covered these many hundreds of arriving at the estimated number of mole- additional molecules it would lose its focus cules, we have not taken into account post- and ability to help scientists classify and translational modifications. Clearly, there is remember them, and the concern that room for error in either direction. But the many reagents were polyclonal and so of conclusion that we are nowhere nearly uncertain specificity. “there” receives some support from two Recognition of the significant capacity other lines of evidence. of intracellular molecules to serve as First, for the recent Eighth HLDA markers of differentiation stage [see, for Workshop, we identified 180 known mole- example, Marafioti et al., (2003)] and that cules that could qualify for CD status, and the workshops could perform a useful task half of them were given CD status at the without necessarily allocating CD names, end of the workshop (Zola et al., 2005). finally led the HLDA Council to include This yield is better than any previous intracellular markers of differentiation HLDA workshop (Fig. 1.1). If we were (Zola et al., 2005), in a set of major changes. nearly at the end of the list, we would expect to be seeing diminishing returns. Second, several proteomics-based dis- 1.1.3 Are We Nearly There? covery projects have found more new mol- Any parent will recognize this question— ecules than known ones, (Peirce et al., 2004; asked by children at any stage in a journey, Nicholson et al., 2005; Loyet et al., 2005; including the very early stages. The child Watarai et al., 2005). Again, there is no evi- has no concept of the length of the journey, dence of the diminishing returns we would so it’s a reasonable question. If the journey expect to see if we had a near-complete of the HLDA is from the early chaos catalog of leukocyte molecules. described earlier in this chapter to the goal The availability of genomic sequence, of having a complete catalog of the leuko- coupled with recent developments in pro- cyte cell surface molecules, how far do we teomics technology, indicates that there is a have still to travel? window of opportunity over the next 5–10 6 LEUKOCYTE MEMBRANE MOLECULES—AN INTRODUCTION Figure 1.1. New CDs assigned per HLDA workshop. The numbers of CD molecules assigned have contin- ued to increase, suggesting that we have not yet characterized the majority of leukocyte membrane molecules. years to complete the discovery process. Thus, given an amino acid sequence, Based on the number of CD molecules usually translated from a DNA sequence, known currently that serve as targets for we can predict the structure, and at least diagnosis and therapy, we can reasonably some likely functions, for any protein.