Immunology Today, voL 8, No. 10, 1987

315Hirashima. M., Yodoi, J. and Ishizaka,K. (1980) J. ImmunoL 125, 1442-1448 36 Rabourdin-Combe,£., Doff, M.E., Guimezanes,A. and Fndman, W.H. (1979) Eur. J. Immunol. 9, 237-242 37 Kiyono, H. Mosteller-Barnum, L.M., Pitts, A.M. eta! (1985) J. Exp. Meal. 161,731-747 38 Yodoi, J., Adachi, M., Teshigawara, K. etaL (1983) J. ImmunoL 131. 303-310 39 Iwata, M., Huff, T.F.and Ishizaka,K. ( ')J. Immunol. 132, 1286-1293 40 Uede,T., Sandberg, K., Bloom, B.R.and Ishiza~ ~ (~983) J. Immunol. 130, 649-654 tt Dresser,D.W. (1978) Nature 274, 480-48 ~. 42 Izui, S., Eisenberg,R.A ~nd Dixon, F.J.(1979)J. Immunol. 122, 2096-2102 43 Van Snik,J. and Coulie, P. (1983) Eur. J. Immunol. 13, 890-894 44 Coulie, P. and Van Snick,J. (1983) Eur. J./mrnunol. 13, 890-894 45 Nemazee,D.A. and Sato, V.L. (1983)J. Exp. Med. 158, 529-545 46 Kissick,W.L (1961)Arthr/t/s Rheum. 4, 4~_4-437 47 Nawato, Y., Koike, T., Hosokawa, H., Tomioka, H. and Yoshida, S. (1985)./. Immunol. 135, 478-482 48 Quinti, I.. Brozek, C., Wood, N., Geha, R.S.and Leun9, D.Y.M. (1986)J. Allergy Clin. Immunol. 77, 586-594 49 Van Snick, J.L and Mason, P.L (1979)J. Exp. Med. 149, 1519-1530

50 Permin, H. and Egeskgold, E.M. (1982)Allergy 37, 421-427 51 Zuraw, B.L, O'Hair, C.H., Vaughan, J.H. etal. (1981)J. Clin. Invest. 68, 1610-1613 52 Van Snick, J.L. (1981)J. Immunol. 126, 815-818 53 Nemazee, D.A. (1985)J. Exp. Med. 161,242-256 54 Nemazee, D.A. and Sato, V.L. (1982)Proc. NatlAcad. 5ci. USA 79, 3828-3832 55 Clarkson,A.B. and Mellow, G.M. (1981)Science214, 186-188 56 Notkins, A.L. (1971)J. Exp. Med. 134, 41s-51s 57 Quinti, I., Brozek, C., Wood, N., Geha, R.S.and Leung, D.Y.M. (1986) J. Allergy C/in. Imnmunol. 77, 586-594 50 Bona, C.A., Finley, S., Waters, S. and Kunkel, H.G. (1982) J. Exp. Med. 156, 986-999 59 Chen, P.P.,Fong, S., Houghten, R.A. and Carson, D.A. (1985)J. Exp. Med. 161,323-331 60 Osmand, A.P., Gewurtz, H. and Friedenson, B.A. (1977) Proc. NatlAcad. Sci. USA 74, 1214-1218 61 Janes, K., Hansen, B. and Gewurz, H. (1981)J. Immunol. 127, 2545-2550 62 Nonaka, M., O'Hair, C.H., Ohno, I. and Katz, D.H. (1986) C/in. Res.34, 671 63 Holmberg, D. and Coutinho, A. (1985)Immunol. Today, 6, 356-357 64 del Guercio, P., Marcelletti, J.F., and Katz, D.H. (1986) Immunol. Today 7, 97-98 65 Pereira,P., Larsson, E-L, Forni, L. etal. (1986)Eur. J. Imnmunol. 16, 685-688

Thesurfaceantigensof humanB lymphocytes During the past few years identification and characterization of the surface antigens of B lymphocytes using monoclonal antibodies has continued apace. !ntema_tiona!W_n.rk~ on. Leucocyte Differentiation Antigens have categorized many antibodies using the cluster of differentiation (CD)system. Here Heddy Zola reviews the recent advances in antigen identification- induding those defined by undustered antibodies as well as CD antigens- and indicates that the bulk of future work will revolve a,mund attempts to place them within a functional framework.

H. Zola

Cells in which the immunoglobulin genes have been rearranged productively are committed B-cell progenitors (abortive rearrangements are found in malignant T cells and even myeloid leukaemic cells). The earliest cells identifiable as B-cell precursors have cytoplasmic immunoglobulin (!~ chain). Once light chain is synthesized, the entire immunoglobulin molecule is expressed on the membrane, the cell is able to recognize antigen and The B-lymphocyte differentiation pathway from commitundergo subsequent antigen-driven differentiation. ted progenitor to plasma cell has been investigated using The production of antibody to a particular antigen monoclonal antibodies to identify markers of differentiadepends on the selective activation of B cells with surface tion on the cell surface. Since this topic was reviewed in immunoglobulin capable of reacting with that antigen. 19831, a few new antigens and many more monoclonal Activation is fo%wed by proliferation (clonal expansion) antibodies have been described. More important, two and immunoglobulin gene mutation, coupled with furmajor advances have strengthened the basis on which ther selection (affinity maturation, class switching). Some the study of B-cell differentiation antigens is built. The cells give rise to antibody-secreting plasma cells, while International Workshops on Leucocyte Differentiation others become memory cells (Fig. 1). This complex Antigens have rationalized the large number of antiprocess does not occur in isolation; the individual steps bodies available into a number of 'clusters of differentiaare controlled by T cells and accessory cells. Hence, the tion', and advances in the study of B-cell funGion have B-lymphocyte membrane must be capable of recognizing enabled questions to be asked about the function of the antigen (native and 'presented'), as well as the various antigens thus identified. regulatory lymphokines. Attempts to correlate known The B-lymphocyte lineage is summarized in Table 1. B-cell membrane molecules with the receptors for these signals provide a fertile area of research - as do lymphokines and the response of B cells to them. Although Departmentof ClinicalImmunology,FlindersMedicalCentre,Bedford these areas are inextricably interwoven, I shall concenPark,Sou~ Australia5042,Australia trate on the B-cell membrane markers. (~ 1987. ElsevierPublications.Cambridge

0167-4919/871502.00

Immunology Today, vol. 8, No. 10, 1987

Experimental approaches The reagents used for s~udies of membrane molecules are now, almost universally, monoclonal antibodies. The ultimate goal is to determine the function of the antigen. Along the way, a number of other questions may be asked. What are the biochemical properties of the antigen? Which antibodies react with the same antigen? How does expression of the antigen correlate with the state of differentiation and activation of B-lineage cells? Biochemical studies have largely been limited to proteins, and restricted to determining the apparent molecular weight, quaternary structure and extent of glycosylation. The reactivity of different antibodies with the same antigen has been the subject of the International Workshops on Leucocyte Differentiation Antigens. While biochemical studies and competitive binding measurements have been a component of these workshops, the major thrust has been a statistical clustering of antibodies which give similar results when tested against a diverse panel of target cells in s~veral different laboratories. This 'clusters of differentiation' system has facilitated rationalization of the large number of antibodies against leucocyte antigens, and clusters of differentiation (CD) are now widely accepted as a means of identifying antigens 2. But clusters can be heterogeneous, and in the most recent workshop, at Oxford, some of the earlier clusters were subdivided. Furthermore, different antibodies in a cluster may have important differences in reactivity, due to differences in epitope specificity, binding affinity and immunoglobulin class. The expression of a particular antigen at different stages within the B-cell lineage is examined by testing the reactivity of the corresponding antibodies against suitable test cells, generally normal cells, malignant cells or cell lines (Table 2). There is clearly a risk of arguing in circles, since the differentiation or activation status of a cell type is based largely on its phenotype. A detailed analysis of the activation/differentiation status of the various cell types is beyond the scope of this review, but it has been discussed extensively elsewhere3-5; similarly, studies of lymphoid tissue can also provide valuable information 6-s but these will not be considered in detail. Emphasis has now shifted to study of antigen function. It is difficult to test directly whether an antibody reacts with the receptor for a particular signal. Antibodies against different molecules may inhibit the same response, whereas antibody against the receptor does not necessarily inhibit the response. Various antibodies may stimulate a cell and thus mimic a physiological ligand, without necessarily being directed against the receptor for that ligand 9. Conversely, antibody against the receptor will not necessarily mimic the ligand. When pure ligand is available, and the receptor is present at

Progression Factors_

•

IL,I

S

\

/

\

--."-~J,~

(@)",

MEMORY B CELL

~,.m~F

YYk

PLASMA BLAST

PLASM& CELL

Fig.1.Activation of mature, unstirnulatedB cells. Antigen and IL-4act together to push res~ B cellsinto the G1 stage of the cellcyde, andindu:e the expressionof receptorsfor 'Drogres~ factors" These factors, which indude B-cell growth factors (BCGFII, low-molecular weig BCGF),and, possibly, IL-2, drive the proliferative cycle. Somecellsemerge from the cell cycle become either memory cells or plasma cells. Differentiation to anYbody-secreting cells stimulated by B-celldifferentiation factor (BCDF).

sufficiently high concentration, the method of choice is ....................... the measurement of binding of the radioactively labelled ligand, and of the inhibition of binding by anti-receptor antibody. Few such studies have as yet been possible with human B-cell molecules. Major B-cellantigens The rest of this review provides brief synopses of the major B-cell antigens (mostly membrane antigens), including those which have been clustered, as well as a few of the more interesting ones which have not. Coverage extends beyond B-lineage specific markers, since B specificity is not always clearly demonstrated, and may not matter (in mice, and possibly also in man, one B-cell growth factor also reacts with eosinophils, and B-cell differentiation factor reacts with mast cells). However, the major histocompatibility complex (MHC) antigens and the transferrin receptor are not discussed. Transferrin receptor expression is associated with cell proliferation, irrespective of differentiation stage, and changes in the expression of the MHC class II antigens as B cell differentiate have been reviewed 1°.11 The expression of the antigens detected by the Blineage-associated clusters of differentiation is summarized in Table 3, while the biochemical properties of the antigens are listed in Table 4. The individual clusters of differentiation are summarized below.

Table 1. Immunoglobulinexpressionand B-celldifferentiation

Cell type Immunoglobulingenes Immunoglobulinexpression

Pluripotential stem cell

Committed progenitor

Germline

VH rearranged Nil

Nil

Pre-B cell

B lymphocyte

Immature/resting Mature/activated VHand VLrearrangedand subjectto mutation

VH transcribed Cytoplasmic1~ MembraneIgM

Maturation independentof antigen

Plasma cell

Membrane IgMIDIG etc.

Cytoplasmic and secreted

Maturationdriven by antigen

309

Immunology Today, vol. 8, No. 10, 1987

!-U Cl)S. The antibodies of the CD5 cluster were originally regarded as T-cell markers (OKT1, Leu 1) but a minor subpopulation of B cells express the 67 000 molecular weight antigen. Most B-lineage chronic lymphocytic leukaemias (CLLs)express the antigen 12, as do a minority of blood and tissue B cells13. CDS-positive B cells appear to be associated with autoimmune disease14.is as are their murine homologues, Lyt-l-positive B cells16. The murine Lyt-1 B-cell subset appears to be a distinct sublineage ~7 capable of unusual combinations of immunoglobulin gene rearrangement and expressiont8. Cl)9. The CD9 cluster was originally defined in the First (Paris) Workshop, by the antibodies BA2, FMC8, DuALL1, SJ9A4 and WB3 (Ref. 19). They react with a protein of apparent molecular weight 24 000 ~p24) which is coded for on chromosome 12 (Ref. 20), and which is expressed by a wide variety of cells including platelets, acute myeloid leukaemic cells21, activated T cells22 and various ce!ls of the B lineage. Acute lymphoblastic leukaemic cells of the B lineage (non-T ALL) express the antigen, as do those of some CLL and more differentiated B-cell malignancies, including pro-lymphocytic leukaemia and multiple myelorna 23. Most B cells in normal blood do not express the p24 antigenz4, so it is not related to differentiation in a simple way. The function of the antigen is being investigated in platelets2s as well as in cells of the B lineage26; its broad yet selective expression suggests that it is involved in an essential cellular process, perhaps one required by platelets continuously but associated only with activation or proliferation in nucleated cells. Some of the antibodies of the CD9 cluster are being used therapeutically, to remove ALt cells from bone marrow for autologous transplantation.

to the B lineage, being found on renal and other epithelia31, occasional T-ALL cells and mature polymorphs. Within the B lineage, some lymphomas and leukaemias of mature phenotype, as well as the pre-B leukaemias, express the antigen, but its function is not known. Monoclonal antibodies against cALLA are being used therapeutically, often in mixtures with CD9 antibodies3Z.33. CD19.This cluster defines a B-lineage-restricted protein of apparent molecular weight 95 000. The antigen was originally defined34 by the antibody B4. Other CD19 antibodies include HD37 (Ref. 35), 4G7 (Ref. 36) and S,~25-C1 (Ref. 37), all of which appear to be directed against the same epitope. The antigen is expressed on 4-8% of blood mononuclear cells, or >90% of blood B cells. Most tissue B cells also express the antigen, although CD19 antibodies give rather weak staining with tissue or blood B cells. In germinal centres of lymph nodes, staining is associated with dendritic cells, but it is not clear whether these cells synthesize the molecule or absorb it from neighbouring B cells. Most B-cell malignancies express the p95 antigen, including B-cellprecursor, chronic lymphocytic leukaemia, and most lymphomas, but not multiple myeloma. Expression of p95 precedes the common ALL antigen. Activation of B cells does not appear to lead to any change in the expression of p95, but CD19 antibodies have been reported to inhibit the response of B cells to co-stimulation by anti-immunoglobulin and interleukin 4 (IL-4), and thi~ immunoglobulin secretion which results from the culture of some B cells with B-cell differentiation factor 38. CD20. The CD20 cluster of antibodies defines a phosphorylated protein of molecular weight 35 000 with little if any attached carbohydrate. The origina~ antibody39 in +hl s.,,, ,.,,,~,'-i"~* . . . .,,,,°~ . . BI, and other antibodies include 2H7 (Ref. 40) and 1F5 (Ref. 41), all of which appear to be directed against the same epitope. Like the p95 CD19 antigen, p35 is essentially restricted to the B-cell lineage, although there is disagreement concerning whether it is found on dendritic cells. Essentially all Ig-bearing B cells express p35, which is thus present on 4-8% of blood

Cl)10. This cluster defines one of the few B-associated antigens - the common acute lymphoblastic antigen (cALLA) - that was well-character~ed before the advent of monoclonal antibodies. Several monoclonal antibodies are now available against this antigen, including J5 (Ref. 27), BA3 (Ref. 28) and VIL-A1 (Ref. 29). The antigen is a single-chain glycoprotein of apparent molecular weight 3o 100 000 and is found on B-cell precursors (including non-T ALL cells). It is not restricted Table 2. Celltypesusedin B-celldifferentiationstudies

Celltype

Normalcells

Pluripotential stemcell Marrowstemcells

Committed progenitor

Pre-B cell

B lymphocyte Immature/restingMature/activated

Marrowcells

BloodB cells

Plasma cell Marrow

Primaryfollicle Germinalcentre I.eukaemiccells

Undifferentiated acuteleukaemia

(7) Celllines

310

ALL: acutelymphoblasticleukaemia CLL: cDroniclymphocyticleukaemia PLL: pro-lymphocyticleukaemia HCL: hairy-cellleukaemia WM: Waidenstrommacroglobulinaemia

CommonALLpre-BALL CMLin lymphoidblast crisis Linesderivedfrom ALL

CLL

PLLHCL

Lymphoblastoidlines

WM

Multiple myeloma 'Plasma cell' lines

Immunology Today, vol. 8, No. tO, 1987

Table3. B-cellmarkersdetectedwith monoclonalantibodies-theB-lineageclustersof differentiation Celltype

Cluster(antigen)

Pluripoten~i~', Committed stemcell progenitor

B lymphocyte Immature/resting Mature/activated

Pre-B cell

Plasma cell

MHCclassII CDg(p24) CD10(CALLA) CDlg (p95) CO20(p35) CD21(p140) CD22(gp130,140) CO23(p45) CO24(p42) CD37(gp40-4S) CO38(gp45) CD39(p80) CDw40(pS0) mononuclear cells, and on B cells in tissues. CD20 antibodies stain B cells strongly. Expression of p35 appears somewhat later than p95 (CD19), and only 50% of non-T ALLs react with CD20 antibodies. With other B-lineage malignancies, CD20 and CD19 reactivity are very similar. The p35 molecule tends to be lost when B cells are activated. CD20 antibodies have both stimulatory and inhibitory effects on B-cell activation, depending on -.he monoclonal antibody used41-43. The major effect of CD20 antibody seems to be to deliver an early signal in activation, taking resting B cells to a stage where they are 'competent' to respond to later signals44. CD21.The antibodies forming the CD21 cluster react with a protein of apparent molecular weight 140 000. The molecule is isolated from the cell membrane as - single chain, and an increase in the apparent molec[,tar weight on reduction 45 suggests intrachain disulphide bonding. The antigen contains N-linked carbohydrate46. The prototype antibody47 was B2, and the antibodies BL-13 (Ref. 48) and HB5 (Ref. 49) reac't, with the same antigen, though not with the same epitope; they react with some T-ALL cells and stain dendritic cells strongly, but are otherwise considered restricted to B cells. CD21 antibodies also stain essentially all B cells in blood and lymphoid tissue, but tissue cells are stained significantly more strongly than blood B cells. In the germinal ce,ltre, the strong staining of dendritic cells makes it difficult to be sure that B cells are being stained, but B cells in the mantle zone stain strongly. The antigen appears somewhat later in B-cell differentiation than the CD19 and CD20 antigens, and most B-cell precursor lines and ALLs are negative. CLLs are positive, while only a proportion of Burkitt's lymphoma react, and myeloma are negative. The p140 molecule has been shown to have two distinct receptor functions - for Epstein-Barr virus and for the complement component C3d (Refs 45,46). The antigen is lost from B cells as they are activated s°, and this loss is more marked than is seen with CD19 and CD20 antibodies. Some of the antibodies comprising the CD21 cluster induce a strong proliferative response, in the presence of T cells9.~1. Polyclonal antibody against the purified p140 molecule enhances the proliferation of B cells in response to B-cell growth factor (BCGF)s2.

CD22.The CD22 cluster was defined during the course of the 2nd (Boston) Workshop by the similar reactivities of the antibodies37 HD6, HD39, 29.110, SJ10-1H11 and SHCL-1. The antibodies react with a pair of highly glycosylated proteins53, precipitating two diffuse bands of apparent molecular weight 130 O00 and 140 090. It is not clear whether both protein chains bear the epitope, or whether they exist as a bimolecular, non-disulphidelinked molecule with the epitope on only one chain. If the protein is deglycosylated, a sharper pattern is obtained, with bands at 100 000 and 85 000. There appear to be a number of distinct epitopes. The antigen is found on most (about 75%) blood B cells but its reactivity with tissue B cells and with malignant cells is complex. In lymph nodes, the mantle zone stains strongly, while the germinal centre stains weakly; dendritic cells ................... Table 4. immunochemicaicharacteristicsof B-ceilantigensdetectedby clusteredantibodies Antigen Prototype Antigen Quaternary GlycosylationReference= antibody molecular structurea (proteincore weight size) CD9

BA2

CD10 CD19

J5 B4

CD20 CD21

B1 B2

CD22

HD39

CD23 CD24

MHM6 BA1

24 000 100000 95 000 35 000 140000 130000/ 140 000 45 000 42 000

Singlechain

Li~leN PossibleO Yes

25,84

Singlechain 30 no internalS-S Singlechain Not known 85-87 Singlechain No 67,85--87 Singlechain N, (120000) 45,46 internalS-S 2 Glycoproteins N, (85 000/ 53,67 no S--S 100000) Possibledimer Yes(43 000) 56,57,63 Singlechain Sialogiyco- 65 orotein Yes(25 000) 67 Singlechain Not known 69 Singlechain Not known 67 Not known Singlechain Not known 44

40-45 000 45 000 CD38 T10 80 000 CD39 G28/8 50 000 CDw40 G28/5 aln most casesthe antigenhas beencharaderizedafter redudion.Its nativefon may havea highermolecularweight. bAdditionalinformationcan be obtainedfrom Ref.67 (in particulara paperby ( Moldenhaueret ai. (B2.2)). 311 CD37

G28/1

....

Immunology Today, vol. 8, No. I0, 1987 ,~

•

are negative. Some pre-B lines are positive, as are a proportion of ALLs. Some ALLs and early B-cell precursorss4 express the antigen only in the cytoplasm. Only about 50% of lymphoblastoid cell lines react, as do 25% of CLLs. Most lymphomas and hairy cell leukaemias react. These findings do not fit with a simple maturationdependent expression, since CLLs are regarded as more mature than ALLs but less mature than hairy cells. In functional experiments, CD22 antibodies enhance the proliferative response of B cells both to antiimmunoglobulin and to growth factor s5. The antigen is lost from cells as they are activated. This cluster of antibodies recognizes a protein of molecular weight 45 000 which i.¢ found on activated but not on resting B cellss6. The observation 57 of an additional broad band at around a molecular weight of 80 000 suggests that the molecule may exist as a dimer in the membrane. Antibodies in the cluster include MHIV16 (Ref. 58), BLAST-2 (Ref. 56) and PL13 (Ref. 37). Deglycosylation of the antigen converts the diffuse 45 000 band to a sharper one at 43 000. Two different epitopes have been identified. B-cell lines- but not pre-B or plasma-cell lines - express the antigen, as do some CLLs and lymphomas. Germinal centre B cells are positive, but mantle zone B cells are not; dendritic cells in the germinal-centre are positive, though they may acquire the antigen from B cells. Recent studies have shown that the p45 molecule recognized by CD23 antibodies serves as the Fc receptor for IgEsg. The antigen appears on B cells after activation, and eviden(~e is mounting that it is involved in the response to BCGFs. Thus, the CD23 antibody MHM6 stimulates the progression of B cells into the proliferative cycle, provided they have received a primary activation stimulus. In this way, the antibody mimics the effect of r'~Jr'u= r t f f i , t¢~ P . / " ~ I ~ ¢ ~ o. uwiu

.I.I,,~ ~ ; . I , , , ~ . ~ . . I.wlx; a l I L l i . P ~ ' U y

: _ 1 - : 1 - : , . - .,,.L~ _ L - - - - - - L I I I I I I U I L ~ LIII~ d U : ~ U r l 3 -

ance of the growth factor by B cells, suggesting that it is directed against the receptor6°-62. It is not yet clear whether the IgE receptor function and interaction with BCGF are related. The CD23 molecule is shed by B cells in the form of a fragment~3 of molecular weight 33 000 which has BCGF-like activity64. This reaction is rapid,

with the half-life of labelled CD23 on the surface being only 1-2 hours, suggesting that the CD23 molecule may have a function in solution. CD24. This cluster consists of antibodies which reaCt6s with a glycoprotein of apparent molecular weight 42 000. The prototype antibody was BA1 (Ref. 66), and HB8 and HB9 (Ref. 37) also belong to this cluster. The antigen is not restricted to cells of the B lineage, being expressed on granulocytes, possibly also on monocytes and on some T-ALLs. The antigen is found throughout the B lineage, although some cell lines and leukaemic samples fail to react. Although differentiated cells of the lineage remain positive, activation is accompanied by loss of reactivity with CD24 antibodies. CB37. This cluster was defined at the 3rd (Oxford) Workshop, and contains the antibodies G28/1, BL14, HD28, HH1, WR17 and F973G6 (Ref. 67) which detect a glycoprotein of apparent molecular weight 40-45 000 with a protein core of 25 000. The antigen is expressed strongly on B cells but is absent from B-cell precursors and from plasma cell lines. There is so far no conclusive evidence of any function for the molecule. CD38. The CD38 cluster was defined at the Oxford Workshop 67, and includes the prototype antibody, TIO (Ref. 68), and HB7 (Ref. 69). Originally studied as a T-cell marker, TIO also reads with plasma cells, germinal centre cells and lymphoid progenitors, but is absent from resting B cells. The antigen has an apparent molecular weight of 45 000. CD39. This is another new cluster defined at the Oxford Workshop, consisting 3t present of two antibodies from the same laboratory, G28/8 and G28/10 (Ref. 67). The antibodies react with a protein of apparent molecular weight 80 000, expressed on B cells and some macrophages, but absent from pre-B cells and plasma cells. One T-cell line and some activated T cells also react with CD39 antibodies.

Table 5, B-cellmarkersdetectedwith unclusteredmonoclonalantibodies

Celltype Antibody(Ref.) FMC1(88) CB2(89) 41H.16(90) BL7(91) L26(77) FMC7(92)

HB4(93) L22(94) L23(94) L24(94)

312

PCA1(75) HH2(95) HB2(96) KB61(97)

Pluripotential stemcell

Committed progenitor

Pre-B cell

B lymphocyte Immature/resting Mature/activated Plasma cell

Immunology Today, voL 8, No. 10, 1987

CDw40. This new cluster contains two antibodies, G28/5 (Ref. 44) and $2C6 (Ref. 67). A third antibody 7o, MA6, was thought to react with the same antigen, but was not fully studied in the Oxford Workshop and so was not included. Unpublished results from my group indicate that it recognizes a different antigen. The CDw40 antibodies recognize a pSO antigen which is found on B cells, carcinoma and interdigitating reticulum cells, but do not react with pre-B cells (which react with MA6) or with plasma cells. The G28/5 antibody shows a strong costimulus with anti-immunoglobulin 71. ClNSR. CD45 refers to a family of glycoproteins known as the 'leucocyte-common antigen' (LCA) or T200. Some determinants on the LCA molecule show restricted distribution, being found only on T cells, only on T-cell subsets or on B cells. Antibodies directed against such restricted determinants were clustered CD45R, while truly leucocyte-common antibodies were clustered as CD45 (Ref. 67). One of the earliest antibodies defining 72 a restricted human LCA determinant was F8-11-13, which is expressed primarily on B cells.

Other dusters.The Oxford Workshop defined three additional clusters, CD30, 31 and CDw32, composed of antibodies which react with various cells including B cells67. CD30 was defined as an activation antigen while CD31 and CDw32 were primarily studied as myeloid antigens. Unclusteredantibodies Many antibodies with interesti ~g reactivity with the B lineage have remained uncluster.~d for various reasons. For instance, a unique antibody cannot be clustered until at least one equivalent antibody is found; some antibodies are not robust enough to pe.rform we!! under workshop conditions; some antibodies simply have not been submitted to, or accepted by, the International Workshops. Some of these antibodies will join existing clusters while some will never be heard of again. Among the unclustered antibodies there are several which are valuable because of their restricted reactivity within the lineage, because they can be used in classifying malignant cells, or because they react with receptors for external signals. Tables 5 and 6 describe a number of monoclonal antibodies which react with B cells over a restricted portion of the maturation pathway, unlike most clusters, which react with much longer pathway segments. Because of their restricted expression, some antibodies are useful in the identification of particular types of leukaemia. FMC7, for example, reacts with a protein of molecular weight 105 000 and has been extensively used in distinguishing prolymphocytic leukaemia and some variants of CLL from the common type. The monoclonai antibody SHCL-2, raised against hairy-cell leukaemia cells73, shows a similar, but not identical reactivity to FMC7 (Ref. 74). Although these antibodies are similar to those of the CD22 cluster, they do show clear differences, including, at least for FMC7, biochemical differences (Tables 4 and 6 and unpublished results). PCA-1 (Ref. 75) and PC-1 (Ref. 76) react with plasma cells, but the staining is generally weak. The CD38 antibodies (such as T10) are not specific to plasma cells and these cells lack good markers. Several antibodies which stain

Table6. Immunochemicalcharacteristicsof B-cellantigens-unclustereo antibodies Antibody

Antigen molecular weight

Quartemary structurea

Glycosylation Reference

FMCl L23 1.24 L26

95 000 295 000 145 000 30 000! 33 000 39 000 105 000 40 000

Singlechain Singlechain Singlechain Twoproteins, not S-Slinked Singlechain Singlechain Not known

Yes Yes Yes Not known

Unpublished 94 94 77

Yes Yes Not known

90 Unpublished 97

41H.16 FMC7 KB61

aln mostcasesthe antigenhasbeencharacterizedafterreduction.Its nativeform mayhavea highermolecularweight. the cytoplasm of plasma cells, and are thus useful in ................. tissue studies, were described in the Oxford Workshop 67. Although most studies on B-cell markers have concentrated on membrane antigens, useful cytoplasmic markers have been described. For example, L26 is a useful marker for B cells in tissue studies77, since it gives strong cytoplasmic staining. Cytoplasmic and membrane expression of an antigen may be seen at different stages of differentiation. Immunoglobulin peptides are a wellknown example, and CD22 antibodies react with the cytoplasm of some B-lineage cells which do not show membrane reactivity53.sa. Since cytcsplasmic antigen is accessible when tissue sections are stained, these results do not always correlate with membrane expression. A particularly interesting group of monoclonal antibodies detects antigens which are absent from resting B cells but appear on activated cells. B5 reacts with a

prnfDin

of = n ~ , ° . ,

m~dlu.-,,l~=-

u=~k÷

-/C ~

...k;.k

appears one day after activation with various stimuli. BAC-1 shows a similar pattern of expression 79, and, without biochemical characterization, it is difficu;t to determine whether it reacts with the same antigen as BS. BB-1 reacts specifically with activated B cells8°, and precipitates a protein of molecular weight 37 000. LB-2, which reacts with a protein of apparent molecular weight 76 000, stains resting B cells; activated B cells show increased expression8°. The antibody anti-Ba reacts with activated B cells81, and inhibits the response of these cells to a BCGF ~ecreted by B cells. The monoclon: dntibodies 4F2 and TROP-4 react with various cells, including monocytes and activated - but not resting - lymphocytes. Suomalainen82 suggests that activated cells returning to the quiescent phase retain the 4F2/TROP-4 marker, while losing other activation markers, such as the transferrin receptor. An antibody designated Y29.33 reacts with malignant, but not normal, B cells in the peripheral blood 83. The corresponding antigen is expressed by normal tissue B cells. Such antigens are difficult to fit into simple maturation pathways, and serve to illustrate the limitations of simple schemes that do not take into account factors such as tissue location and cell-cycle stage.

Future prospects The B-cell membrane expresses a number of glycoprotein molecules which are involved in the complex interactions between B cells and other components of the

313

Immunology Today, voL 8, No. 10, 1987

immune system. As B cells mature from committed progenitor to plasma cell by way of several cycles of activation and proliferation, the display of surface structures changes. Monoclonal antibodies provide powerful probes to identify, quantify, isolate and analyse surface markers. At present, two areas of study - function and phenotype - are just beginning to meet. A number of functional receptors clearly exist, and we can identify a number of proteins on B cells using monoclonal antibodies. Soon we may know which proteins mediate what function. I ~m indebted to Mrs Mary Brown and Mr Alan Bentley for preparing the typescript and diagram, respectively, and to the Anti-Cancer Foundation of the Universities of South Australia, which supported my participation in the International Workshops on Leucocyte Differentiation Antigens.

1 McKenzie, I.F.C. and Zola, H. (1983) ImmunoL Today4, 10-15

2 Bernard, A., Bemstein, I., Boumsell, L. etal. (1984)Disease Markers 2,443-446 3 Greaves, M.F., Delia, D., Robinson, J., Sutherland, R. and Newman, R. ( | 981) Blood Cells 7, 257-280 4 Zola, H., McNamara, P.J., Moore, H.A. etal. (1983) Clin. Exp. Immunol. 52, 655-664 $ Anderson, K.C., Bates, M.P., Slaughenhoupt, B.L. etal. (1984) B/ood 63, 1424-1433 6 Hsu, S-M. and Jaffe, E.S.(1984)Am. J. Pathol. 114, 387-395 7 Bhan, A.K., Nadler, L.M., Stashenko, P., McCluskey, R.T. and Schlossman, S.F.(1981)1 Exp Med. 154, 737-749 8 Hofman, FM., Yanagihara, E., Byrne, B etaL (1983)Blood 62, 775-783 9 Clark, E.A. and Ledbetter, J.A. (1986) ImmunoL Today 7, 267-270 10 Guy. K. and van Heyningen, V (1983) ImmunoL Today4, 186-189 . . . .

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12 Royston, I., Majda, J., Baird, S., Meserve, B. and Griffiths, J. (1980)/Immunol. 125, 725-731 13 Ledbetter, J.A. and Clark, EA. (1986) Hum. !mmunol. 15, 30-43 14 Plater-Zyberk,C., Maini, R.N., Lam, K., Kennedy, T.D and Janossy, G. (1985) Arthritis Rheum. 28, 971-976 15 Sowden, J.A., Roberts-Thomson, P.J.and Zola, H. (1987) Rheum. Int. (in press) t6 Hayakawa, K., Hardy, R.R., Herzenberg, L.A. and Herzenberg, L.A. (1983)Prog. ImmunoL 5, 661 -665 17 Hayakawa, K., Hardy, R.R., Herzenberg, L.A. and Herzenberg, L.A. (1985) J. Exp. Med. 161, 1554--1568 18 Hardy, R.R., Dangl, J.L., Hayakawa, K. eta/. (1986)Proc Natl Acad. Sci. USA 83, 1438-1442 19 Bernard, A., Boumsell, L., Dausset,J., Milstein, C. and Schlossman, S.F.(eds) (1984) Leukocyte Typing, SpringerVerlag 20 Katz, F., Povey, S., Parkar, M. etal. (1983) Eur. J. Immunol. 13, 1008-1013 21 Ashman, L.K., White, D., Zola, H. and Dart, G.W. (1987) Leukaemia Res. 11, 97-101 22 Hercend, T., Nadler, L.M., Pesando,J.M. etal. (1981)Cell. Imrnunol. 64, 192-199 23 San Miguel, J.F., Caballerro, M.D., Gonzalez, M., Zola, H. and Borrasca,A.L (1986)Br. J. HaematoL 62, 75-83 24 Zola, H., Moore, H.A., McNamara, P.J.etal. (1984)DL~,~c Markers 2, 399-417 25 Gorman, D.J., Castaldi, P.A., Zola, H. and Berndt, M.C. (1985) Nouv. Rev. Fr. Hematol. 27, 255-259

26 Zipf, T.F., Antoun, G.R., Lauzon, G.J. and Longenecker, B.M. (1986) in Leucocyte Typing II (Reinherz, E.L., Haynes, B.F., Nadler, L.M. and Bernstein, I.D., eds), pp. 203-211, SpringerVerlag 27 Ritz, J. Pesando,J.M., Notis-McConarty, J., Lazarus, H. and Schlossman, S.F.(1980)Nature 283, 583-585 28 LeBien, T.W., Bradley, J.G., Boue, D.R. etal. (1984)in Leucocyte Typing (Bernard, A., Boumsell, L., Dausset, J., Milstein, C. and Schlossman, S.F., eds), pp. 346-353, SpringerVerlag 29 Knapp, W., Majdic, O., Bettelheim, P. and Liszka, K. (1982) Leukaemia Res. 6, 137-147 30 Newman, R.A., Sutherland, R. and Greaves, M.F. (1981)J. Immunol. 126, 2024-2030 31 Metzgar, R.S., Borowitz, M.J., Jones, N.H. and Dowell, B.L. (1981)J. Exp. Med. 154, 1249-1254 32 Ramsay, N. et al. (1985)Blood, 66, 508-513 33 Bradstock, K.F., Favaloro, E.J., Kabral, A. etal. (1986) Pathology 18, 197-205 34 Nadler, L.M., Anderson, K.C., Marti, G. etal. (1983) J. Irnmunol. 131,244-250 35 Moldenhauer, G., Dorken, B., Schwartz, R. etal. (1986) in Leucocyte Typing I/(Reinherz, E.L., Haynes, B.F., Nadler, L.M.

and Bernstein, I.D., eds), pp. 61-67, Springer-Verlag 36 Meeker, T.C., Miller, R.A., Link, M.P., Bindl, J., Warnke, R. and Levy, R. (1984) Hybridoma 3, 305-319 37 Nadler, L.M. (1986)in Leucocyte Typingll, (Reinherz, E.L., Haynes, B.F., Nadler, L.M. and Bernstein, I.D., eds), pp. 3-43, Springer-Verlag 38 Pezzutto, A., Dorken, B., Rabinovitch, P.S.etal. (1987) in Leucocyte Typing III (McMichael, A.J. et al., eds), Oxford University Press(in press) 39 Stashenko, P., Nadler, L.M., Hardy, R. and Schlossman, S.F. (1980)J. Immunol. 125, 1678-1685 40 Clark, E.A. and Yokochi, T. (1984)in Leucocyte Typing (Bernard, A., Boumsell, L., Dausset, J., Milstein, C. and Schlossman, S.F.,eds), pp. 339-346, Springer-Verlag 41 Clark, E.A., Shu, G. and Ledbetter, J.A. (1985) Proc Natl Acad. Sci. USA 82, 1766-1770 42 Golay, J.T., Clark, E.A. and Beverley, P.C. (1985)J. Immunol. 1,3:3,

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43 Tedder, T.F., Boyd, A.W., Freedman, A.S., Nadler, L.M. and Schlossman, S.F.(1985) J. Irnmunol. 135, 973-979 44 Clark, E.A. and Ledbetter, J.A. (1986) Proc NatlAcad. Sci. USA 83, 4494-4498 45 lida, K., Nadler, L. and Nussenweig, V. (1983)J. Exp. Med. 158, 1021-1033 46 Shaw, M.F.E., Nemerow, G.R. and Cooper, N.R. (1986) J. Immunol. 136, 4146-4151 47 Nadler, LM., Stashenko, P., Hardy, R. etal. (1981)J. Immunol. 126, 1941-1947 48 8rochier, J., Magaud, J.P., Cordier, G. et al. (1984)Ann. ImmunoL (Inst. Pasteur) 135D, 283-299 49 Tedder, T.F., Clement, L.T. and Cooper, M.D. (1984) J. Immunol. 133, 678-683 50 Boyd, A.W., Anderson, K.C., Freedman, A.S. etal. (1985)J. Immunol. 134, 1516-1523 51 Nemerow, G.R., McNaughton, M.E. and Cooper, N.R. (1985) J. Immunol. 135, 3068-3073 52 Frade, R., Crevon, M.C., Barel, M. etal. (1985)Eur. J. Immunol. 15, 73-76 53 Dorken, B., Moldenhauer, A., Pezzutto, A. etal. (1986) J. Immunol. 136, 4470-4475 54 Dorken, B., Pezzutto, M, Kohler, IVi., Thiel, E. and Hunstein, W. (1987)in Leucocyte Typing III (McMichael, A.J. etal., eds), Oxford University Press(in press) 55 Pezzutto, A., Dorken, B., Moldenhauer, G. and Clark, E.A. (1987)J. Immunol. 138, 98-103 56 Thorley-Lavvson, D.A., Nadler, L.M., Bhan, A.K. and Schooley, R.T. (1985) J. Immunol. 134, 3007-3012 57 Ravoet, A-M. and Lebacq-Verheyden, A-M (1986)

Immunology Today,vol. 8, No. 10, 1987 ..........

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Leucocyte Typing II(Reinherz, E.L., Haynes, 8.F., Nadler, L.M. and Bernstein, I.D., eds), pp. 213-225, Springer-Verlag 58 Rowe, M., Hildreth, J.E.K., Rickinson, A.B. and Epstein, MA. (1982) Int. J. Cancer 29, 373-381 5S Kikutani, H., Inui, S., Sato, R. etal. (1986) Cell 47, 657-665 60 Gordon, J., Webb, A.J., Walker, L., Guy, G.R. and Rowe, M. (1986) Eur. J. Immunol. 16, 1627-1630 61 Gordon, J., Webb, A.J., Guy, G.R. and Walker, L. (1987) Immunology 60, 517-521 62 Gordon, J., Webb, A.J., Walker, L, Guy, G.R. and Rowe, M. (1987) in Leucocyte Typing III (McMichael, A.J. et al., eds), Oxford University Press(in press) 63 Thorley-Lawson, D.A., Swendeman, S.L. and Edson, C.M (1986)J. Imrnunol. 136, 1745-1751 64 Swendeman, S.L. quoted in Immunology 60, 517-521 65 Pirruccello, S.J. and LeBien, T.W. (1986) J. Immunol. ! 36, 3779-3792 66 Abramson, C.S., Kersey, J.H. and LeBien, T.W. (1981) J. Irnmunol. 126, 83-88 67 McMichael, A.J. et al. (eds) Leucocyte Typing III, Oxford University Press(in press) 68 Reinherz, E.L., Kung, P.C., Goldstein, G., Levey, R.H. and Schlossman, S.F. (1980)Proc. NatlAcad. Sci. USA 77, 15881592 6S Tedder, T.F., Clement, LT. and Cooper, M.D. (1984) Tissue Antigen 24, 140-149 70 Chan, K.H., Yip, T.C., Ng, W.L and Ng, M.H. (1985) 1nt. J. Cancer 36, 329-336 71 Shields, J.G., Smith, S.H. and Callard, R.E. (1987) in Leucocyte Typing III (McMichael, A.J. et al., eds), Oxford University Press(in press) 72 Dalchau, R. and Fabre, J.W. (1981)J. Exp. Med. 153, 753-765 73 Posnett, D.N., Chiorazzi, N. and Kunkel, H.G. (1982) J. Clin. l~est. 70, 254-261 74 Robinson, D.S.F., Posnett, D.N., Zola, H. and Catovsky, D. (1985) Leukaemia Res. 9, 335-348 75 Anderson, K.C., Park, E.K., Bates, M.P. etal. (1983) J. Immunoi. 130, 1132-1138 76 . . . . . . . . . . . . ,.., Bates, M.P., Siaughenhoupt, B., Schlossman, S.F. and Nadler, L.M. (1984)J. Irnmunol. 132, 3172-3179 77 Ishii, Y., Takami, T., Yuasa, H., Takei, T. and Kikuchi, K. (1984) Clin. Exp. Immunol. 58, 183-192

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78 Freedman, A.S., Boyd, A.W., Anderson, K.C. et al. (1985)J. Immunol. 134, 2228-2235 79 Suzuki, T., Sanders, S.K., Butler, J.L. et al. (1986)J. Immunol. 137, 1208-1213 80 Clark, E.A., Ledbetter, J.A., Holly, R.C., Dinndorf, P.A. and Shu, G. (1986)Hum. Immunol. 16, 100-113 81 Kikutani, H., Kimura, R., Nakamura, H. etal. (1986)J. Immunol. 136, 4019-4026 Suomalainen, H.A. (1986) J. ImmunoL 137, 422-427 83 Hirt, A., Baumgartner, C., Forster, H.K., Imbach, P. and Wagner, H.P. (1983) Cancer Res. 43, 4483-4485 84 Newman, R.A., Sutherland, D.R., LeBien, T.W., Kersey, J.H. and Greaves, M.F. (1982)Biochim. Biophys. Acta 701, 318-327 85 LeBien, T.W., Bradley, J.G., Platt, J.L. and Pirruccello, S.J. (1986) in Leucocyte Typing II (Reinherz, E.L, Haynes, B.F., Nadler, L.M. and Bernstein, I.D., eds), pp. 169-175, SpringerVerlag 86 Clark, E.A. and Einfeld, D. (1986)in Leucocyte Typing II (Reinherz, E.L., Haynes, B.F., Nadler, L.M. and Bernstein, I.D., eds), pp. 155-167, Springer-Verlag 87 Horibe, K. and Knowles, R.W. (1986) in Leucocyte Typing II (Reinherz, E.L., Haynes, B.F., Nadler, L.M. and Bernstein, I.D., eds), pp. 187-201, Springer-Verlag 88 Brooks, D.A., Beckman, I., Bradley, J. etal. (1980) Clin. Exp. Immunol. 39, 477-485 • J Jephthah, J., Terasaki, P.I., Hofman, F. etal. (1984)Blood 63, 319-325 90 Zipf, T.F., Lauzon, G.J. and Longenecker, B.M. (1983)J. Immunol. 131, 3064-3072 91 AI-Katib, A., Wang, C-Y. and Koziner, B. (1985) CancerRes. 45, 3058-3063 92 Brooks, D.A., Beckman, I.G.R., Bradley, J. etal. (1981)J. Immuno/. 126, 1373-1377 93 Tedder, T.F., Clement, L.T. and Cooper, M.D. (1985) J. Immunol. 134, 1539-1544 94 Takami, T., Ishii, Y., Yuasa, H. and Kikuchi, K. (1985)J. Immunol. 134, 828-834 95 Smeland, E., Funderud, S., Ruud, E., Blomhoff. H.K. and Godai, T. (1985) Stand. J. ImmunoL 21,205-214 96 Abo, T., Landay, A., Balch, C.M. and Cooper, M.D. (1985) Hum. Imrnunol. 13, 253-264 97 Pulford, K., Ralfkiaer, E., Macdonald, S.M etal. (1986) Immunology 57, 71-76

therapy. Two perceptions of 'food allergy' were delineated recently by Charles May: 'orthodox' and 'unby Jonathan Brustoff and Stephen J. Challa- orthodox' (J. Allergy Clin. immunol. combe, Bailliere Tindall, 1987. £75 (x+ 1016 (1986) 78, 144). The orthodox view pages) ISBN0 7020 11568 holds that a few well-defined symptom complexes (e.g. anaphylaxis, Handbookof FoodAllergies urticaria, asthma, coeliac disease, by James C. Breneman, Marcel Dekker Inc., etc.) are the result of known im1987. $59.75 (US and Canada)$71.50 (else- munological mechanisms to foods, and have been associated with food where) (x + 278 pages)ISBN0 8247 7558 9 hypersensitivity responses in adeWith the increasing use of well- quately controlled studies. The undesigned placebo-controlled clinical orthodox view perceives the orthotrials in the past 10 years, immuno- dox viewpoint as representing only a logic, metabolic and pharmacologic fraction of the often ill-defined reactions to foods are being clearly symptoms resulting from food ingesdefined. Despite these advances, tion. Such symptoms are sald to there remains considerable con- result in a variety of behavioral, troversy regarding symptoms in- psychiatric, neurologic and somatic duced by adverse food reactions and complaints. Not unexpectedly, diappropriate means of diagnosis and agnostic and therapeutic approaches

FoodAllergyand Intolerance

....

differ substantially between advocates of the two viewpoints. In their encyclopedic review of adverse food reactions, Brostoff and Challacombe have intentionally included both viewpoints. As stated in the preface, contributions from clinicians and scientists were sought in order 'to help understand the immunopathological and other processes in our patients. Their points of view are diverse . . . There is no suggestion that, because we have invited particular authors to contribute to our book, we necessarily agree with their view. Occasionally the reverse is true!' Unfortunately, the editors provide little guidance to the uninitiated or casual readeJ, which could result in acceptance of some of the scientifically unsubstantiated views presented. Unless

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