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 nologists came to appreciate the power of all reacted with the same . The monoclonal antibodies as reagents, large differences in the individual descriptions numbers of new antibodies were made, reflected differences in technique, 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 , and non-T-non-B gens (HLDA), organized in Paris by Lau- acute lymphoblastic . 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 , 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 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 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 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 “”); 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 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 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 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, 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 . 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. expect to find many new diagnostic and Structural prediction is most effectively therapeutic targets among the molecules to done with Web-based resources, which will be discovered. be described in Chapter 2. It is important to remember that such structures, and derived functions, are predictions and will 1.2 STRUCTURE AND FUNCTION almost certainly be wrong in some aspect. The functional requirements of immune Most molecules that serve as useful cells are diverse, and the molecules that markers of differentiation on leukocyte mediate surface interactions are corre- membranes are . There is spondingly diverse in structure. Neverthe- enormous variation of structure, but less, several molecular themes recur, and there are also recognizable themes and classifying the molecules according to their motifs. Structural features of proteins are molecular structure is helpful in under- summarized in Boxes 1.1 to 1.4. Function standing both structure and function. depends on structure, so structure can predict function to a significant degree. 1.2.1 Protein Domains Determination of the three-dimensional structure of a molecule depends on X-ray Protein domains (see Box 1.1) are distinct diffraction analysis or nuclear magnetic subunits of proteins that are associated resonance studies, which are complex and with a particular molecular function, such require time and resources. Only a few as protein–protein interactions or kinase leukocyte membrane molecules have their activity. One characteristic of domains, structure analyzed at this level, but as the which is used in identifying them, is that number of “solved” structures increases, interactions between residues within a our ability to predict the tertiary structure domain are generally stronger than inter- of proteins from amino acid sequence actions between residues in different improves. domains. Domains usually occur as individ- STRUCTURE AND FUNCTION 7

Box 1.1. TERMS USED IN DESCRIBING STRUCTURAL FEATURES OF PROTEINS

Domain: A protein domain is a distinct Motif: Motif is used to describe a subunit of a protein. Many proteins are shorter sequence than a domain or composed of several domains, separated module, but a sequence associated with a by short or long sequences that may act particular structural or functional feature. as “hinge” regions or “spacers.” Domains Family: Proteins are grouped together have characteristic structural features, in families based on structural similari- and about 20 domains have been ties. Because the structural features that described (Table 1.1). Domain structure allow the family relationship to be dis- dictates domain function. It is believed cerned are largely located in the domains, that there are domain structures still to be and many proteins have multiple differ- discovered. ent domains, a protein may be assigned to Module: The term “module” can be several families. The term “family” is used interchangeably with domain. It applied to the gene as well as the protein. tends to be used when describing a Superfamily: Superfamilies are domain in a protein that has several dif- broader groupings that include several ferent domains—a modular protein. families that show similarity.

ual regions of a protein sequence that have Differences in the intracellular domains a defined three-dimensional structure. can result in proteins with identical or Domains may be present in both the extra- similar extracellular domains having cellular and the intracellular regions of a opposite functions. For example, the four protein. Extracellular domains are likely to TRAIL receptors (CD261, CD262, CD263, be involved in interactions with other cells, and CD264) that are encoded by separate extracellular matrix or extracellular signals, genes all have a single extracellular TNFR whereas intracellular domains are likely to domain, but they differ by the presence, be involved in signaling to trigger the cel- absence, or truncation of an intracellular lular response to the extracellular interac- Death domain. tions. The association of a domain with a The sequencing of the human genome particular function is useful in assigning has lead to the development of programs probable cellular functions to otherwise that can annotate conserved protein poorly characterized proteins. domains automatically. These annotations At least 20 functional domain types have can be used to assign some function to pro- been identified within the CD molecules teins that have not been identified using (Table 1.1). Individual proteins may have antibodies. The InterPro database (www. multiple copies of a single domain, for ebi.ac.uk/interpro) (Mulder et al., 2005) example, CD22, which has five adjacent provides a single access resource for the Ig-like domains, and CD30 (TNFRSF8), major protein domain signature databases. which has three consecutive TNFR domains, or they may contain a mixture of 1.2.2 Protein Families different domains, for example, CD62L, which contains c-type , EGF, and The classification of proteins into families sushi (short complement-like repeat) originally relied on the presence of a domains. shared protein domain and could therefore 8

TABLE 1.1. Protein domains found commonly in leukocyte surface molecules Domain Structure Functions Example CD Molecules ABC Transporter Multiple transmembrane domains coupled to nucleotide ATP-dependent transport of substances CD243, CD338 binding domain across membranes A beta-sandwich formed of seven strands in two sheets. Ca++ Adhesion CD144, CD324, CD325 ions bind residues from neighboring domains. C-type Lectin Open structure consisting of five beta strands and two alpha Calcium-dependent sugar binding CD62L, CD72, CD141 helices, with four loops of relatively unstructured sequence. CD205, CD303 CUB Ig-like beta barrel with four conserved cysteines that Complement components CD304 probably form two disulphide bridges (C1ÐC2, C3ÐC4). DEATH A homotypic protein interaction module composed of a Regulation of apoptosis and inflammation CD95, CD261, CD271 bundle of six alpha-helices. through the activation of caspases and NF-kappaB EGF A two-stranded beta-sheet followed by a loop to a C- Unknown CD62L, CD97, CD141, terminal short two-stranded sheet. The domain includes six CD339 cysteine residues that have been shown to be involved in disulphide bonds. 2 The domain contains four conserved cysteines involved in Protein binding CD205, CD206, CD222 disulphide bonds and is part of the collagen-binding region of fibronectin. Fibronectin 3 Seven beta-strands modeled to fold into antiparallel beta- Protein binding CD122, CD130, CD171 sandwiches with a topology that is similar to immunoglobulin constant domains. GPCR Seven-transmembrane helices, G-protein binding loop. Receptors CD97, CD195, CD294 Ig-like The fold consists of a beta-sandwich formed of seven strands in ProteinÐprotein and proteinÐligand CD3, CD19, CD50, CD80, two sheets, usually stabilized by intradomain disulfide bonds. interactions CD171, CD226, CD300a LDLR Complement-like cysteine-rich repeats Bind multiple ligands CD91 Link Two alpha helices and two antiparallel beta sheets arranged Hyaluronan(HA)-binding region CD44 around a large hydrophobic core similar to that of C-type lectin. LRR 2Ð45 motifs of 20Ð30 amino acids in length that generally ProteinÐprotein interactions CD180, CD281 folds into an arc or horseshoe shape. Protein tyrosine Cytoplasmic protein kinases. Catalyses the addition of a phosphate CD117, CD136, CD167a, kinase group to a tyrosine residue CD331 SCR Domain contains four repeats of a well-conserved region, which Protein binding CD5, CD6, CD163 spans 115 amino acids, and contains six conserved cysteines Semaphorin A variation of the beta propeller topology, with seven blades Detect and respond to chemical CD100, CD108, CD136 radially arranged around a central axis. Each blade contains signals a four-stranded (strands A to D) antiparallel beta sheet. Sushi The structure is based on a beta-sandwich arrangement; one Protein binding CD21, CD25, CD35, face made up of three beta-strands hydrogen-bonded to CD62L form a triple-stranded region at its center and the other face formed from two separate beta-strands. TNF Central beta sheet. Trimeric CD153, CD154, CD178 TNFR Repeats containing six conserved cysteines, all of which are Binding of TNF domains CD30, CD40, CD95 involved in intrachain disulphide bonds. Phosphotyrosine Cytoplasmic tyrosine-specific protein phosphatases. Catalyzes the removal of a phosphate CD45, CD148 phosphatase group attached to a tyrosine residue 9 10 LEUKOCYTE MEMBRANE MOLECULES—AN INTRODUCTION

Box 1.2. CARBOHYDRATE STRUCTURES

Carbohydrates may be attached to pro- The monosaccharides that occur most tein () or lipid (glycolipid). often in leukocyte antigens are as follows:

Glucose (Glc) Galactose (Gal) Mannose (Man)

N-acetyl galactosamine (GalNac) (N-acetyl neuraminic acid)

Monosaccharides are linked together alpha or beta. In the example given, it is to form oligosaccharides or polysac- the alpha orientation. charides. Glycosamino glycans (GAGs) are Each linkage is described as follows: polysaccharides that are chains of disac- Glc-alpha1-3GalNac means glucose is charides or longer oligosaccharides that linked from its number 1 carbon to carbon include an amino sugar.The amino sugar is 3 of N-acetyl galactosamine. The forma- generally glucosamine or galactosamine, tion of the link changes the #1 carbon of and it is often acetylated or sulfated. The the glucose from a state that allows free major GAGs found in leukocyte mem- rotation to a state in which it is fixed in brane molecules are chondroitin sulfate one of two mirror-image orientations and or . STRUCTURE AND FUNCTION 11

Box 1.3. MEMBRANE ATTACHMENT AND ORIENTATION OF PROTEINS

Integral membrane proteins cross the Type IV integral membrane proteins lipid bilayer. In the simplest form, an are multipass proteins, like type III pro- integral membrane protein consists of an teins. However, in type IV proteins, the extracellular region, a membrane span- proteins form a hydrophilic pore through ning region, and a cytoplasmic region.The the membrane. membrane spanning region is generally Type V refers to proteins that do not an alpha helix consisting largely of pass through the membrane but are hydrophobic amino acids, allowing it to attached to it by their C-terminus via gly- be stable in the hydrophobic lipid cosyl-phosphatidylinositol (a GPI link). environment. Some proteins include a In more detail, the C-terminal amino acid charged residue in the membrane- is linked to ethanolamine, which is in turn spanning region, which interacts with linked through a phosphodiester link to charged residues on other proteins. a short string of sugars that link via Type I integral membrane proteins myoinostol through a phosphodiester to have the N-terminus outside the cell and glycerol and thence to the membrane the C terminus in the cytoplasm. A signal fatty acids. sequence is cleaved from the N terminus In addition, proteins may be linked to before export to the cell surface. the membrane through other proteins, via Type II integral membrane proteins salt bridges, disulphide bonds, or non- have their C terminus outside the cell and covalent interactions. the N-terminus in the (generally short) Proteins that do not pass through the cytoplasmic sequence. The membrane- membrane, or have a short cytoplasmic spanning sequence is uncleaved but sequence devoid of signal-initiating resembles a signal sequence for protein motifs, may nevertheless transducer secretion. signals to cells through interaction with Type III integral membrane proteins other proteins that do have signaling pass the membrane several times. Exam- capacity.Receptors often consist of two or ples are found ranging from two to seven more protein chains, where one binds the passes through the membrane. ligand and the other transmits the signal.

group proteins that appear very distinct. the assignment to a family is usually based The largest family that includes CD mole- on a single domain that can be associated cules is the immunoglobulin superfamily, with a common protein function. There which includes proteins such as CD3, have been recent efforts to formalize the CD19, CD80, CD90, and CD200. Other assignment of protein to families, based on large families that include multiple leuko- aspects of the evolutionary relationship cyte cell surface antigens are the TNF and between proteins (Wu et al., 2004). TNFR superfamilies, the C-type lectin The members of those families that are superfamily, and the tetraspanins (Table more closely related tend to occur in clus- 1.2). Even though many leukocyte cell ters in the genome, such as the KIR family surface proteins contain different domains, of immunoreceptors, which are located on 12

TABLE 1.2. Some protein families that have CD molecules as members Family Domain Functions Example CD Molecules ADAMs Metallopeptidase Matrix degradation CD158a, CD158c Cadherin Adhesion CD144, CD324, CD325 IgSF Ig-like, ICAM ProteinÐprotein and proteinÐligand CD3, CD19, CD80, CD171, CD226, CD300a, interactions CD50, CD242 Heterodimer of alpha and beta chains Adhesion CD18, CD41, CD61, CD49a, CD103 LILR Ig-like Immunoreceptors CD85-family, CD335 MS4A 4-transmembrane domain Various CD20 Rhodopsin GPCR 7-transmembrane domain Receptors CD191, CD193, CD294 Secretin GPCR 7-transmembrane domain Receptors CD97 Lectin, EGF, sushi Adhesion CD62l, CD62E, CD62P SEMA Semaphorin, plexin Cell migration CD100, CD108, Sialic acid-recognizing Ig-superfamily Adhesion CD22, CD33, CD170 Tetraspanins Four transmembrane domain Various CD9, CD37, CD81, CD151 TNFRSF TNFR Binding of TNF ligand domains CD27, CD30, CD40, CD95, CD271 TNFSF TNF Trimeric cytokines CD70, CD153, CD154, CD178, CD252 Note: The domains listed are characteristic domains of the families. The functions are indicative of a function for the family. UTILITY IN RESEARCH, DIAGNOSIS, AND THERAPY 13

Box 1.4. POST-TRANSLATIONAL PROTEIN MODIFICATION

Carbohydrate attachment to protein is Proteins may be sulfated, at tyrosine, either through asparagine (N-linked gly- serine, or threonine. cosylation) or through the OH of serine Covalent acylation affects protein or threonine (O-linked glycosylation). compartmentalization and function. Pro- Asparagine is glycosylated only if the teins may be acylated as follows: sequence is Asn-X-Ser or Asn-X-Thr, but not if X is Pro. Even with the right neigh- • Palmitoylation through thioester boring amino acids, gycosylation is not linkage to (usually) cysteine residues automatic and may vary for the same • Myristoylation through amide linkage protein depending on the tissue and other to the N terminal glycine conditions, so that these sequences are referred to as potential N-glycosylation • Prenylation through thioether linkage sites. to C terminal cysteine O-glycosylation can occur at any Ser or • Glypiation through phophatidyli- Thr, but it is more likely in regions rich in nositol linked to the C-terminal amino Ser, Thr, and Pro. acid after removal of the signal are proteins linked to sequence glyosylaminoglycans (GAGs) (see Box 1.2). The GAGs are generally linked to Palmitoylation and glypiation affect pre- protein through serine or threonine. dominantly membrane proteins and Glycosylation varies from tissue to direct them into lipid raft regions. tissue and between stages of activation Proteins are frequently cleaved into and differentiation. two or more components by proteases Proteins can be phosphorylated either after synthesis and before they can be at tyrosine or at serine and threonine, expressed or function. A common which occurs generally in the cytoplasmic example is the removal of a signal part of the molecule. Phosphorylation (by sequence in proteins expressed with the kinases) or dephosphorylation (by phos- N terminus outside the cell. When pro- phatases) is often the first stage of a sig- teins are cleaved, the fragments may stay naling cascade leading to activation of together, held by disulphide bonds or by gene transcription. noncovalent interactions. in humans, or the MS4A et al., 2001) has changed the world irre- family of which CD20 is a member, which versibly. As with all significant knowledge, is located on chromosome 11. Other fami- once we have it, we cannot revert to not lies can be spread throughout the genome: having it, and if we ignore it, we risk be- There are members of the Ig-like super- coming irrelevant. However, the euphoria family on each chromosome. that accompanied the publication of the genome sequence was quickly followed by a reassessment of what we still need to 1.3 UTILITY IN RESEARCH, know, to make use of the genomic infor- DIAGNOSIS, AND THERAPY mation. Even before completion of the sequence, those involved were turning The availability of the full human genome their attention back to function and hence sequence (Venter et al., 2001; McPhersen to proteins (Broder and Venter, 2000), 14 LEUKOCYTE MEMBRANE MOLECULES—AN INTRODUCTION the rapidly developing field known as chemical methods, used together with proteomics. video image analysis, allow studies of Knowing the DNA sequence is a start- tissue sections, which approach flow ing point. From the genome we need to cytometry in precision and sensitivity, define the genes—the coding sequences. and additionally provide information on Every cell has the same genes (ignoring spatial relationships. Several for the moment processes where parts of formats provide sensitive and precise the genome are spliced out or mutated methods of measuring the concentration of during the course of differentiation). What molecules in solution, provided specific characterizes different cells with different antibodies are available. functions is the set of genes that is The ability of antibodies to act as ago- expressed. So the emphasis shifts from nists or antagonists facilitates the analysis gene sequencing to understanding the of the functions of molecules, particularly protein profile of cells and how it relates to those on the cell surface. Finally, antibodies function. can make therapeutic reagents, for The sequence information is being con- example, in cancer (Grillo-Lopez et al., verted rapidly into a catalog of genes, which 1999) and in situations where we need to can be searched for molecules of likely rel- modulate immune reactivity (Nashan et al., evance to the immune system in a variety 1997). of ways. We can look for homologs to known human immunoreceptors or, more broadly, for members of molecular families 1.3.1 Major Applications that are known to be used by the immune of Antibodies system. From the sequence information, we can construct probes and use Northern CD markers provide an outstanding blots, in situ hybridization, microarray example of the power of antibodies in iden- technology, or polymerase chain reaction tification, quantitation, and localization of (PCR) to determine what is being cells in order to analyze function and expressed in cells of the immune system disease state. (See Table 1.3 for major appli- and what is differentially expressed in cations). using multiple different cells or states of the immune antibodies conjugated to different fluores- system. cent dyes, beads, or nanoparticles allows the Antibodies remain uniquely useful tools determination of cell lineage and sublin- in the analysis of expression and function eage using several different markers in of molecules, particularly surface mem- widely available instruments. For example, brane molecules. Although methods based T cells are identified by the expression of on mRNA detection are improving in CD3, and the T cells of the “helper” subset quantitative precision and accuracy, there (which interact with antigen-presenting is no direct and universal relationship cells using MHC Class II to present antigen) between the amount of mRNA for a are identified by expression of CD4.Within protein in the cell and the amount of the CD3/CD4 double-positive subpopula- protein actually present, and it is the tion, antigen-experienced (memory) cells protein that mediates the function. Flow are identified by the expression of CD45R0 cytometry has sophistication, precision, and can be further subdivided into effector dynamic range, and ability to analyze on memory and central memory cells using one a single-cell basis that will be difficult to of several markers.This example of multipa- improve on, and it works particularly well rameter analysis uses four antibodies and with antibodies as probes. Immunohisto- colors, in addition to two physical parame- REFERENCES 15

TABLE 1.3. Outline of applications of antibodies against leukocyte markers Application Feature Example Identification Multiparameter, multiplex Flow cytometric cell-by-cell analysis Antibody microarray Localization Multiparameter Immunohistology Quantitation Multiplex Cytometric bead array, ELISA Isolation/purification Multiparameter Cell sorting, protein purification Therapy Specificity, potency Transplantation and cancer therapy—e.g., CD3 and CD20 antibodies

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