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Monoclonal antibodies to B cells
hnmunologl' Today, z,ol. l, No. l, 198d
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1 M o n o c l o n a l antibodies to B cells Ian F. C. McKenzie and Heddy Zola Monoclonal antibodies ( Mab) have deJined fimclional T lymphoo, te subsets in mouse and man: L y - 1 / O K T d positive helper T cells, Ly- 19,,]/OKT8 positive kdler T (ells and Ly-2d/OKT8 positive suppressor T cells. An equi~,alent array q/-use~d reagents for B-cell subsets is nol ),et available - allhough many laboratorie.~ are trying to produce lhem. ht this" review lan Mekenzze and Heddy Zola summarize the Mab which are m,adable and which detecl polrmorpbic or monomorphtc determinanls on the B-cell surface in both man and mouse. T h e definition of functional T-cell subsets has been one of the most productive a n d useful exercises of the past decade. Progress in defining B-cell s u b p o p u l a tions has been slower but m a y be q u i c k e n e d by the use of M a b . We i n t e n d to review the success M a b have received so far but will exclude antibodies to surface (s) a n d cytoplasmic (c) i m m u n o g l o b u l i n (Ig), a n d Fc or c o m p l e m e n t receptors, a l t h o u g h it is clear that Bcelt subsets can now be defined in the mouse on the basis of the relative a n d absolute a m o u n t s of I g M and IgD present (Herzenberg, L. A. - personal comm u n i c a t i o n ) . M a n y of the M a b we will describe do not react exclusively with B cells - b u t this does not deny their usefulness, in the same way that a n t i - T h y - 1 anti-' bodies, not entirely T-cell specific, have b e e n powerful in the detection of T cells in the mouse. T h e p a t h w a y ( s ) taken by B cells as they differentiate from pluri-potential stem cells t h r o u g h i n t e r m e d i a t e stages to the t e r m i n a l p l a s m a cell stage has been m a p p e d in some detail, using i m m u n o g l o b u l i n expression as the principal marker, a n d since i m m u n o g l o b u l i n is the principal functional product of B cells, this is appropriate. T h e differentiation has been considered as m a t u r a t i o n along a single p a t h w a y a n d evidence for the differentiation of B cells into divergent pathways, differing in function ( c o m p a r a b l e to the T 4 a n d T8 p a t h w a y s of the T cells) is not compelling. However, there m a y be considerable heterogeneity w i t h i n the c u r r e n t l y defined m a t u r a t i o n stages, a n d M a b should help to i n t r o d u c e m o r e d e t a i l into the ' m a p ' , p a r t i c u l a r l y as they can be used to purify subp o p u l a t i o n s for functional analysis, e n a b l i n g a definition relating p h e n o t y p e to function, as has been done for T cells. Monoclonal
a n t i b o d i e s to m u r i n e B c e l l a n t i g e n s
la antigens. A large n u m b e r of a n t i - I a M a b have been described ~ 4 ( T a b l e I) a n d several s u m m a r y statements can be made: (i) M a b recognize existing specificities (la.2, 3, 7, 8, 9, 15, 20, etc.) or new specificities; (ii) the M a b confirm a n d extend the existing knowledge of la defined by alloantisera, i.e., they detect 1-A or I-E encoded specificities; (iii) one Department of Pathology, University of Melbourne, Parkville, Victoria, Australia; and Department of Clinical Immunology, Flinders Medical Centre, Bedford Park, South Australia. e Elscxwr Blotnedl~M Pres, 1983 0167-4tH0/83/[11)1)0~001~/$1
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interesting a n t i b o d y ~ (CE 197) detects a cross reaction b e t w e e n I-A;, a n d l-Eh- subregions; (iv) several M a b ~',= to the ' h y b r i d ' Ia.22 a n t i g e n have been described (this a n t i g e n is present in strains of H-2 haplotype k, absent in b a n d d, b u t present in ((b x d)F I mice); (v) on the basis of sensitivity to pronase a n d resistance to n e u r a m i n i d a s e a n d glycosidases, (protein - p) or the reciprocal ( c a r b o h y d r a t e - c) M a b detect two different classes of Ia molecules s lap a n d laC; e.g. several antiIa.2 or Ia.17 M a b a p p e a r to recognize the same specificity, yet one detects laP, the other IaC; (vi) the rat a n t i - I a a n t i b o d y M R C O X 6 a p p e a r s to react exclusively with the I-A [3 c h a i n '~ suggesting that the 13 chain is more i m p o r t a n t in p o l y m o r p h i s m t h a n the a chain. While of great interest in studies of i m m u n e function, a n t i - l a sera are of limited value in disc r i m i n a t i n g b e t w e e n B-cell subsets in the mouse as they are present on all or most B cells, but a b s e n t from stem cells a n d the early stages of B-cell differentiation 1°. TABLE I. Monoclonal anti-murine B-cell antibodies Designation
Reaction
Reference
A. Ia
I-A (la. 2,3,7, 8, 9, 15, stem ,clg ,slg ,PFC + 20 etc.) I-E (Ia. 7, 22) I-A and I-E (CE197) la. 22 ('hybrid') la t,, la, (la. 2, 17) MRC OX c' ([3chain)
1-4 1-4 5 6, 7 8 9
B. B-cell specific
Lyb-2.1, 2.3 Ly-m20.2 (Ly-17.2) PC-2
stem , clg +, sIg +, PFCstem?, early BY, slg +, PFC ~ B cells , PFC'
11-15 16 18
C. Rat antl-mouse
DNL 1.9, RA3-2C2, 14.Stem , cIg ÷, slg +, PFC ~ 19B5 (ATA) stem , clg +, sIg +, PFC?
10, 19, 20 26, 27
D. Potentially useful B-cell + T-cell Mab
ThB Qa-m2 LY-ml8 Ly-m19 J1 ld
(50% thymus, all B) (10% thymus, 100% T, 15%B) (15% thymus, 90% T, 80% B) (90% thymus, 50% T, 50% B) (90% thymus, 0% T, most B)
22 23 24 25 28
Immunology To&O',v,l. d, .#b. I, I(.]8.3 L~cb-2.1. The Lyb-2 locus was originally defined by conventional antisera ~t but more recently ~2 by M a b to the Lyb-2.1- specifities2.L Lyb-2 is situated on chromosome 4 and in contrast to other l:y loci has. three alleles. The M a b react with a molecule of mol. wt 45,000 which is present only on B cells, and on their Ig- precursors, but not on plasma cells. It has been suggested that the Lyb-2 site on B cells may be involved in the generation of plaque forming cells (PFC). t3-1s, Ly-m20.2. This antigen has recently been described by a series of cytotoxic M a b (Ref. 16) produced as A anti-70Z/3 or (A x C 3 H / A n ) F I anti-70Z/3 (70Z/3 is a pre-B cell tumor). Ly-m20.2 is present on B cells in spleen, lymph node; on 50% of bone marrow cells, but is not on thymus cells. Ly-m20.2 is p r o b a b l y present on all B cells as the bone marrow reaction is high and I g M - P F C were positive. The Ly-m20 locus is linked to Mls on chromosome is probably the same as Ly-17 iT, and could be related to the Mls or Ly-M loci. Like many of the murine Ly specificities which are not lymphocyte specific, Ly-m20.2 is found on liver and kidney.
PC.2. The plasma cell PC.1 alloantigenic system was defined some years ago by conventional alloantisera, and in attempts to make M a b , the PC.2 locus was described TM after a fusion of the spleen cells of ( D B A / 2 x C 5 7 B L / 6 ) F I mice immunized with the M O P C 70A plasmacytoma. PC.2 is found on several plasmacytomas and also appears to be present on both IgM and IgG PFC of all strains. No M a b to PC. 1 have been described. T h e M a b d e s c r i b e d above were m a d e after mouse/mouse immunizations and fusions and with the exception of PC.2, recognize polymorphic antigens. The next series are xenogeneic (rat anti-mouse) immunizations and recognize non-polymorphic or monomorphic determinants.
D N L 1.9. A M a b was produced i,~ by immunizing rats with 70Z/2 and detects an antigen present on all cells of the B-cell but not T-cell lineage, including sIg , cIg + cells, and both IgM and IgG PFC. In addition, a proportion of s I g - and c I g - cells were D N L 1.9 +. This is an interesting antibody as spleen colony-forming units (CFU-S) and culture colony-forming units (CFU-C) are D N L 1.9 , and with the finding of I g DNL 1.9 + cells an early B-cell precursor may have been defined.
RA
~-2C2. A n o t h e r M a b was p r o d u c e d 2° by immunizing rats with the Abelson murine leukemia virus-induced pre-B cell line R A W 112. The antibody is similar in reaction to D N L 1.9 and both M a b could recognize the same determinant. RA 3-2C2 +, I g - cells can give rise to slg + cells in vitro within 2 days, thus confirming the detection of early B cells by these two antibodies. It has also been demonstrated that a proportion of the R A 3-2C2 + I g - cells in spleen and lymph node are also Ly-2 + (S. Morse - personal communication). A similar antigen to these could be B2A2
11 which is absent from C F U - S but present on most B cells including the ' w e - p r o g e n i t o r cells '21.
14.8. Another series of rat M a b anti-murine pre-B cell tumors have been described and it is likely that the previous antibody and some of these M a b are similar. Of interest is the finding that several of this series (177.17 and 83.4) react with h u m a n cells. In addition, the 14.8 M a b reacts with a glycoprotein of mol. wt 220,000 which is the B-cell equivalent of the T-200 molecule and has also been defined in man. Extensive studies on the time of a p p e a r a n c e of antigens indicate that 19B5 (see below) appears before 14.8 and Lyb-2 antigens, both of which a p p e a r before Ia antigens in the mouse. Another series of M a b detect antigens on both T and B cells but could be useful in studying purified Bcell subpopulations. These are: ThB. The ThB locus was defined 22 by a M a b produced after a fusion using a rat spleen immunized with MOPC-104E. T h e antigen is present on all B cells, not on T cells and on 50% of thymus cells. O f interest is the finding that strains are high or low with regard to expression of ThB on B cells and the gene controlling the phenomenon is linked to Ly-6. W h e t h e r ThyB is present on early or late B cells (PFC) has not been reported. Qa-m2. The Q a series of loci (Qal-5) map to the right of the H-2 complex and are found on all T cells and a p r o p o r t i o n of B cells. T h e Q a - m 2 m o n o c l o n a l antibody recognizes a polymorphic determinant (M F 39,000 and [32M) found on all T cells, < 10% of thymus cells and only 10-15% of B cells > . It is not clear at present whether early B cells are Q a - m 2 +, but lgm PFC are Qa-m2- and IgG PFC are Q a - m 2 +. In purified B-cell populations this M a b may be useful in differentiation of the stages in B cell development. L y - m l S , L y - m l g . These antigens have recently been described 24.> and could be of interest as they react with 50% and 90% of Ia + B cells, respectively. The L y - m l 8 M a b was made against spleen cells and Ly-m 19 against the 70Z/3 tumor.
A TA (Abeison - target associated antigen). The 19B5 M a b was made by immunizing rats with mouse brain 2~', and reacts with thymus (98%), bone marrow (35%), spleen (34% - where ~17% of both T and B cells react) but not with lymph node cells. In bone marrow, the C F U - S are negative but the target cell for Abelson virus infection - presumably an early B cell is eliminated. In addition, several pre-B cell tumors are positive (e.g. 70Z/3), as are B cells in fetal liver. The B-cell regenerative capacity of bone marrow cells is impaired after treatment with the 19B5 M a b (Ref. 27).
J l l d . The J1 ld M a b reacts with red cells, thymus (not mature T cells) and with most B cells 2~, although it can distinguish B-cell subsets because primary IgM responses but not secondary IgG responses can be ablated. T h e M a b d e s c r i b e d thus p a r t l y d i s c r i m i n a t e
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hnmunologv Today, vol. t, No. 1, 1983
between the different stages of B-cell differentiation b u t most are present on all or most B cells. However, the PC.2 a n t i g e n is confined to plasma cells, Lyb-2 is absent from these cells a n d the D N L 1.9, RA3-2C2 a n d 14.8 a n t i b o d i e s recognize the precursor of the c I g sIg- B cell. T h e a p p r o a c h for the p r o d u c t i o n of B-cell M a b m a d e so far has been to either i m m u n i z e with whole cells from spleen etc., or with B cells a n d hope for the best, or to use t u m o r s such as 7 0 Z / 3 which are regarded as ' p r e - B ' cells a n d to test on these t u m o r s or a panel of t u m o r s 29,3°used to classify B-cell differentiation steps. U n f o r t u n a t e l y , m a n y of these cells also carry m a r k e r s which represent later stages of B-cell development.
TABLE It. Monoclonal antibodies reacting with human B lymphocytes.
Monoclonal
B.
a n t i b o d i e s to h u m a n B c e l l s
Designation Reaction A.
Antibodies against 'Ia-like' molecules
Many described, including:DA2 la (p28, 33) 2.06 la (p28, 34) SI.9; S1.5 Ia (p28, 33) FMC 4 Ia (p28, 33) L203; L227 la (p28, 34) SG157 and I-E (p29, 34) and l-A (p30, 33), SGI71 respectively FMC 2 HLA-DRw4 Genox 3.53 HLA-DR-I, 2, 6 (supertypic) MC 26 IR-new
FMC 1 FMC 7 BI (p30) B2 (p 140)
Ia ( H L 4 - D R ) . A large n u m b e r of M a b reacting with m e m b r a n e 'Ia-like' a n t i g e n s have b e e n p r o d u c e d (Table II) a n d although analysis of the specific gene products detected by different M a b is incomplete, a n u m b e r of interesting features are emerging. Most of the M a b described react with n o n - p o l y m o r p h i c determ i n a n t s , but some react with H L A / D R associated polymorphic antigens. A n example is F M C 2 ~7 which reacted with some, but not all H L A - D R w 4 donors. Another, MC-26, defines a new D R specificity u n r e l a t e d to a n y k n o w n specificity TM. M o r e c o m m o n l y , p o l y m o r p h i c d e t e r m i n a n t s d e t e c t e d have b e e n ' s u p e r t y p i c ' , e.g. Genox 3.533~, which reacts with H L A - D R w 1, 2 a n d 6. T h e antibodies against n o n - p o l y m o r p h i c d e t e r m i n a n t s show considerable heterogeneity, a n d different M a b a p p e a r to detect several different gene products. For e x a m p l e L203 a n d L227 detect two different p o p u l a t i o n s of p28,32 molecules on the cell line Raji >. Such M a b will e n a b l e the identification of gene products equivalent to the m u r i n e 1-,4 a n d I - E subregions. M a n y of the antibodies described thus far p r o b a b l y identify I-E-like antigens, whilst antibodies SG 157 a n d SG 17 ! identify I-E-like a n d l-A-like molecules, respectively 36. A further d i m e n s i o n in the heterogeneity of gene products of the I region is the description of carbohydrate-defined as well as polypeptide-defined polym o r p h i s m s similar to that found in the mouse (Betts, R. L. a n d M c K e n z i e , 1. F. C. - m a n u s c r i p t in p r e p a r a tion). As only one or two M a b have b e e n described thus far which define k n o w n D R specificities, it is unlikely that equivalent antisera to the h u m a n antiD R sera will be easily produced, but the value of the existing M a b for tissue t y p i n g has yet to be determined.
Y29.55
M a t u r e B cells. M a b s reacting with B cells in blood or secondary l y m p h o i d tissue have been described (b, T a b l e II) a n d have been of some diagnostic value. For example, F M C 1 has been used in e n u m e r a t i n g ' B cells in the diagnostic l a b o r a t o r y for 2 years 3'~, a n d generally parallels sIg as a B-cell marker, whereas
Tul Ab89 H76
37 31 38
All B cells in blood 39 B cell subpopulation 40 All B cells in blood 42 Weak reaction with blood B cells but 43 strong reaction with tissue B cells Tissue B cells and B leukaemias/ 44 lymphomas, but only small proportion of normal blood B cells Sub-population of tissue B cells 45 No reaction detected with normal B cells 46 Murine antigen cross-reactive with 47 human, probably MHC product
Antibodies which are not restricted to the B-cell lineage but may provide a useful classification within the B lineage.
F8-1 l- 13 (p215) PI 153/3 BA/1 FMC 3 FMC 8 VIC-Y1 D.
31 32 33 34 35 36
Antibodies reacting only with B cells (in haemopoietic tissue)
A n u m b e r of M a b which react with h u m a n B cells have been described ( T a b l e II) which includes antibodies which are not B-cell specific b u t nevertheless show selective reactivity w i t h i n the B-cell lineage.
C.
Reference
B-cell-associated determinant of 48 leucocyte-common antigen. Strong reaction with B cells, weak with T cells. Neuroblastoma, B cells, non T-non 49 BALL. B cells, pre-B ALL, granulocytes 50 Some B cells, only some B-CLL. Most 51 T cells, monocytes. Some B cells, only some B-CLL; 52 Platelets, monocytes, granulocytes. Cytoplasmic marker on B cells and 53 monocytes.
Antibodies reacting with possible precursors of B lymphocytes
J5 (gp95) c-ALL, some normal marrow cells. VIL-A1 c-ALL, some normal marrow cells. BA/2 (p24) c-ALL, some normal marrow cells.
54 55 57
F M C 7 reacts with a B-cell s u b p o p u l a t i o n 4" a n d is useful in distinguishing p r o l y m p h o c y t i c leukaemia from chronic lymphocytic l e u k a e m i a 41. M a b B1 a n d B2 show a n interesting difference in their reactivity with blood B cells a n d B cells in l y m p h nodes 42,43. M a b Y29.55 is absent from most blood B cells, b u t is expressed on tissue B cells a n d on the m a l i g n a n t cells of B leukaemias a n d l y m p h o m a s 44. T u l shows a n even more restricted distribution, being found almost exclusively on germinal centre B cells a n d some B-cell m a l i g n a n c i e s 4~. Ab89 reacts with a limited range of Bl y m p h o m a s a n d no reactivity with n o r m a l B cells has been detected 46. M a b H76 has not been studied extensively in m a n ; it is a n a n t i b o d y p r o d u c e d against mouse B cells which cross-reacts with a n H L A - l i n k e d a n t i g e n on h u m a n B cells, possibly H L A - D R or a related p r o d u c t 47.
Immunology Today, vol. -f, No. 1, 19,~¢3
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A n t i b o d i e s reacting w i t h B and n o n - B cells. T h e antibodies listed in Table IIc are not specific for B cells, but nevertheless provide interesting information on the classification and maturation of B cells. M a b F8-11-13 reacts with a p215 antigen which is the leucocyte-common antigen detected by other M a b and it also reacts strongly with B cells, but weakly or not at all with other leucocytes, indicating that it reacts with a B-cell associated determinant found on some molecules of the leucocyte-common a n t i g e n 4s. This relationship of F8-11-13 to the leucocytecommon antigen is of interest and suggests that differentiation may be accompanied by changes in limited parts of molecules common to m a n y related cells a striking distinction the concept of differentiation markers as from whole molecules, their presence depending on the differentiation pathway taken by the cell. PI 153/3, a M a b p r o d u c e d a g a i n s t neuroblastoma cells, also stains B cells a n d pre-B leukaemia cells 49. The BA/1 Nlab reacts with B cells, acute lymphoblastic leukaemia (ALL) cells of pre-B type, but also reacts with granulocytes~L F M C 3 reacts with some B and T cells and with chronic lymphocytic leukaemia (CLL) cells in only a minority of cases, providing information on the heterogeneity found in this disease <. F M C 8 similarly distinguishes between B-cell subpopulations, but also reacts with platelets, monocytes and granulocytes s2. Vic-Y1 is a cytoplasmic marker found in B cells and monocytes, and serves as a reminder that specific markers are not
to be found exclusively at the cell membrane~L B cell precursors. In addition to the foregoing, some important M a b react not with mature B cells, but with their precursors (Table II-d). J5 and Vii-A1 Mab react with C A L L A antigen, previously defined by rabbit antisera ~4`s'. (This marker is present on ALL cells of the ' c o m m o n ' type, c-ALL; it is also present on a small n u m b e r of cells in normal marrow, but is nevertheless very useful in the diagnosis of leukaemia). M a b BA/2 reacts with a marker which has a similar but not identical expression 57. M a b a n d B cell m a t u r a t i o n In T a b l e I I I a simplified B-cell maturation scheme is used as a framework on which to compare the monoclonal antibodies. Cells differentiating from the progenitor stern cell are considered to be committed to the B lineage when rearrangements of immunoglobulin (Ig) genes have been initiated, since these are characteristic of the B lineage. Although there is no simple test to identify such committed progenitors, pre-B cells can be readily identified by the expression of low levels of cytoplasmic immunoglobulin (cIg). Pre-B cells mature into B cells, which have detectable surface m e m b r a n e i m m u n o g l o b u l i n (slg) but no readily-detectable cIg. These sIg + cells are the B lymphocytes of the blood and secondary lymphoid tissues and are heterogeneous in terms of amount and class of slg. Finally B cells can be stimulated to
TABLE III. B-cellmaturation a Stem c e l l A.
B.
C.
pre-B
early B
late B
Plasma cell
clg +
slg +
slg ++
clg "4-+ secreted Ig
Immnnoglobulin characteristics Germ-line genes
lg genes re-arranged
Occurrence in normal blood/tissue Bone marrow
Bone marrow
Bone m a r r o w
CommonALL
pre B-ALL
Blood/secondary lymphoidorgans
Germinal centres
Leukaemia model
Null leukaemia
D.
B-committed Progenitor
CLL
PL; HCLa Muhiple myeloma Waldenstrom's macroglobulaemia
Monoclonal antibody reactivity
Anti la (p28, 33) PI 153/3 FMC 8 (p24) B1 B2 BAI BA2 (p24) J5 (gp 100) VIL-A1 FMC 1 FMC 7 FMC 3 apLL and HCL: prolymphocytic and hairy cell leukaemia; data drawn from references in Table II, 41, 59-61, and Zola el al (unpublished). Dotted lines ...... areas of particular uncertainty; . . . . . reaction with a proportion of cells; - a definitereaction.
Immunology Today, vol..'I, .¥a. 1, 1983
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differentiate into plasma cells, the functional end cells of the B lineage. In relating phenotype to position in the differentiation pathway normal cells (b, Table III), leukaemic cells (c, Table III) or lymphoblastoid cell lines may be used, although there are considerable uncertainties in relating a leukaemic cell or cell line type to a position in the normal maturation pathway. In particular, the use of cALL + c l g - leukaemias as representatives of the B-committed progenitor is based on the observation s" that Ig gene rearrangements can be detected in cALL but not in T.ALL. The data in Table III reinforce the idea that cALL + cells are early members of the B lineage. A preliminary assignment of the reactivities of many of the Mab is shown (Table IV-d). Bearing in mind the heterogeneity of the data (obtained from studies of leukaemic cells, cell lines, and on normal cells) the assignment must be tentative. The forthcoming International Workshop on H u m a n Leucocyte Differentiation Antigens should clarify the reactivities of these antibodies. It will be noted that several Mab detect antigens present on most of the B lineage, but none encompass the entire maturation sequence ([3-2 microglobulin and HLA do appear throughout the lineage, and on most other nucleated cells). One antigen (detected by F M C 3) seems unrelated to maturation, being found on a proportion of cells at all stages. Several of the antibodies listed show a different reactivity with B cells of lymphoid organs and B cells in blood. Thus, T u l and Y29-55 are absent from normal circulating B cells but present on tissue B cells and circulating leukaemic B cells. B2 reacts strongly with sessile B cells in lymphoid tissue but weakly with circulating B cells. Furthermore, maturation from primary to secondary follicles is accompanied by changes in the relative expression of B1 and B2 (Ref. 59). None of the antibodies described so far is restricted to stem cells or the B-committed progenitor. This probably reflects the screening panels that have been used to detect Mab, and the use of new tumors, screening on a panel of tumors or performing functional screening may provide the answer. For example, M. Beveridge (personal communication) is assaying by the CFU-S technique - keeping the hybrid colonies growing for 7 days until the results are available. In addition, it will be necessary to detect reactivity with extremely small (<1-5%) proportions of bone marrow cells because precursors may only b e present in such small numbers (note: none of the markers which define T-cell subsets in the mouse are present on <10% of cells.) Clearly more Mab are needed and given these, our understanding of B-cell differentiation and function should increase greatly o v e r the next few y e a r s , Acknowledgment The studies reported herein were supported by funds obtained from the National Health and Medical Research Council of Australia.
References 1 Lemke, H., Hammerling, G. J., Hammerling, U. (1981) lmmunoL Rev. 47, 175-206 2 0 i . V T., Jones, P. P, Goding, J. W., Herzenberg, L. A. and Herzenberg, L. A. (1978) Curr. Top. Mu'robwl, lmmunol. 81, 115-129 3 0 z a t o , K., Mayer, N. and Sachs, 1). H. (1980).)'. bnmumd. 124,533-54(I 4 Klein, J., Huang, H. J. S., Lemke, H., Hammerling, G. J. and Hammerling, U. (1979) bnmlmogenetm~8, 419-432 5 Symington, F. W. and Sprent, J. (1981) lmmum,genetlc~ 14, 53-61 6 Sachs, D. H., Mayer, N. and Ozato, K. (1981) Re~. Monogr. lmm.nol. 3, 95-101
7 Harris, J. F. and Delovitch, T. L. (1980).7. lmman,,I. 125, 2167-2176 8 Higgins, T. J., Parish, C. R , Hogarth, P. M., McKenzie. I. F. C. and Hammer[ing, G . J . (1980) hnmunogenettc, 11. 467-482 9 McMaster, W. R. (1981 ) lmrmmogenetu.~ 13,347-350 10 Kincade, P. W. (1981) Adv. hnmunol. 31,177-245 I I Sato, H., Boyse, E. A. (t976) bnmunogenetu'~ 3, 565-572 12 Shen, F. W. (1981) Rer. Monogr. bnmunol. 3, 25-31 13 Yakura, H., Shen, F. W., Boyse, E. A and Tang, L. (1980) Immuno~,'netu~ 10. 603-605 14 Yakura, H., Shen, F. W., Kaemmer, M. and Boyse, E. A. (1981) ,7" Exp. Med. 153, 129-135 15 Kincade, P. W., Lee, G., Scheld, M. P. and Blum, M. D. (1980) J. lmmunol. 124, 947-953 16 Kimura, S., Tada, N , Nakayama, E., Liu, Y., Hammerling, U. (1981) lmmunogenetu'~ 14, 3-14 17 Shen, F. W. and Boyse, E. A. (1980) lmmunogenetu s 11, 315-317 18 Tada, N., Kimura, S., Hoffman, 1%'1.and Hammerling, U. (1980) bmmmogenehc~ 11, 351-302 19 Dessner, 1). S., Loken, M. R. (1981) Ear.,7. lmm.nol. 11,282-285 20 Coffman, R. L. and Weissman, 1 L. (1981).7. Exp. Med. 153,269-279 21 Layton, J. E., Baker, J., Bartlett, P. F. and Shortman, K. (1981) .7. lmmunol. 126, 1227-1233 22 Eckhardt, L. A., and Herzenberg, L. A. (1980) lm,mmr~enel.~ 11, 275-291 23 Hogarth. P. M.. Crewther, P. A and McKenzie, 1. F. C. Eur. ,7. lmm.nol. (in press) 24 Kimura, S., TadA, N., Liu, Y. and Hammerling, U. (1981) lmmum,genetu'~ 13, 547-554 25 Tada, N., Kimura, S., Liu, Y., Taylor, B. A. and Hammerling, U. (1981 ) lrnmunogenetu~ 13, 539-546 26 Shinefield, L. A., Sato, V. L., and Rosenberg, N. E. (1980) Cell. 20, 11-17 27 Paige, C . J . , Kincade, P. W., Shinefield, L. A. and Salo, V. L. (1981) J. Exp. Med. 153, 154-165 28 Bruce, J., Symingtom, F. W., McKearn, T. J., and Sprent, J. (1981) J. lmmunol. 127, 2496-2501 29 Warner, N. L., Daley, M. J., Richey, J. and Spellman, C. (1979) Immuno. Rev. 48, 197-243 30 Lanier, L. L., Warner, N. L., Ledbetter, J. A., Herzenberg, L. A (1981) J. lmm.nol 127, 1691-1697 31 Brodsky, F., Parham, P., Barnstable, C.J. et al. (1979) lmmunol Ree. 47, 3-61 32 Charron, D. J. and McDevitt, H. O. (1979) 1%~. ,Natl Acad. ,qc,. U.S.A. 76, 6567-6571 33 Trueco, M. M , Garotta, G., Stocker, J W. and Ceppellini, R. (1979) lmmuno/qgual Roy. 47, 219-252 34 Beckman, 1. (;, R., Bradley, J., Brooks, D. A., Kupa, A., McNamara, P.J., Thomas, M. and Zola, tt. (1980) Chn. E~p. lmmunol 40, 593-601 35 Lampson, L. A. and Levy, R. (1980).)'. bnm.nol. 125, 293-299 36 Goyert, S. M. and Silver, J. (1981).Vature¢ London) 294, 266-268 37 Zola, H., Bradley, J., Macardle, P., McEvoy, R. and Thomas, Miriam, (1980) Tran~plantat,,n 29, 72-73 38 Thompson, C. H., Vaughan, H. A. and McKenzie, 1. F. C. (1982) lhcman hnnmnol. (submitted for publication) 39 Brooks, D. A., Beckman, I., Bradley, J., McNamara, P. J., Thomas. M. E. and Zola, H. (1980) Clin. Exp. lmmunol. 39, 477-485 40 Brooks, D. A., Beckman, 1. G. R., Bradley, J., McNamara, P. J., Thomas, M. E. and Zola, H. (1981).7- Immunol. 126, 1373-1377 41 Catovsky, I)., Cherchi, M , Brooks, 1)., Bradley,J. and Zola, H. (1981) Blood 58, 406-408 42 Stashenko, P., Nadler, t,. M., Hardy, R. and Schlossman, S. F. (1980) J. lmmunol. 125, 1678-1685 43 Nadler, U M., Stashenko, P., Hardy, R., Agthoven, A., Terhorst, C. and Schlossman, S. (1981).7 bnmumd 126, 1941-1947 44 Gudat. F. G , Forster, H. K , Girard, M. F.. Albrecht, R , Ludwig, C. and Obrecht, J. P. (1981) in Leukaemza Mar£ers (W. Knapp. ed) pp. 109-112. Academic Press, London
Immunology Today, vol. 1, .No. 1, 1983 45 Ziegler, A., Stein, H., Muller, C. and Wernet, P. (1981) in Leukaemm Marker, (W, Knapp, ed.) pp. 113-116, Academic Press, London 46 Nadler, L. M., Stashenko, P., Hardy, R. and Schlossman, S. F. (1980) .7. lmmumd. 125, 570-577 47 Gasser, l). L., Winters, B A., Haas, J. B., McKearn, T. J. and Kennett, R H, (1979) l:'n,~..Vall A, ad. S,I. U.&A. 76, 4636~.640 48 I)alchau, R. and Fabre, J, (1981).7. E~p. Med. 153,753-765 . 49 (;reaves, M. F., Verbi, W,, Kemshead, J. and Kennett, R. (1981/) Blood 56, 1141-1144 51") Abramson, G, S., Kersey, J. H. and LeBien, T. W. (1981),7. hnmunol. 126, 83-88 51 Zola, H., Beckman, I. (L R., Bradley, J., Brooks, D A., Kupa, A., McNamara, P. J., Smart, I. J and Thomas, M. E. (1980) hnmunohJgy 40, 143-150 52 Brooks, 1) A . Bradley,J. and Zola, H. (1982) Pathology, 14, 5-I 1 53 Majdic, O , Sfingl, G , Liszka, K., Honigsmann, H. and Knapp, W. (1981) in Leulaemm Marker, (W. Knapp, ed.) pp. 125-127, Academic Press, London
15 54 Ritz, J., Pesando, J. N1, Notis-McConarty, J., Lazarus, H and Schlossman, S. F. (1980).~hture( London; 283, 583-585 55 Liszka, K , Majdic, O., Bettelheim, P. and Knapp, W. (1981) in Leukaemm MarL,'r~ (W. Knapp, ed.) pp. 61-64, Academic Press, London 56 Brown, (;., Capellaro, D. and Greaves, M. F. (1975)J. ,~1/()m~er In~t. 55, 1281 57 Kersey, J. H., LeBien, T W., Abramson, C. S., Newman, R,, Sutherland, R. and (;reaves, M. ( 1981 ) .7- E~.7~..lied. 153,726-731 58 Korsmeyer, S. J., Hieter, P. A., Ravetch, J. V , Poplack, D. G., Leder, P. and Waldmann, T. A. (1981) in Leukaemta Mar~er~ (W. Knapp, ed ) pp. 85-97, Academic Press, London 59 Bhan, A. K., Nadler, L. M., Stashenko, P, McCluskey, R. T. and Schlossman, S. F. (1981 ) ,7. E,:p. Med. 154, 737-749 60 Nadler, L. M., Ritz, J., Reinherz, L. and Schlossman, S. F. (1981)in Leukaemta Mar~er~ (W. Knapp, ed. ) pp 3-17, Academic Press, London 61 Kersey, J. H., Le Bien, T. W., Gajl-Peczalska, K., Nesbit, M., Jansen, "F., Kung, P., Goldstein, G., Sather, H., Coccia, P., Siegel, S., Bleyer, A, and Hammond, D. (1981) in Leukaemia Markers (W. Knapp, ed) pp. 453-462, Academic Press, London
10
1 M o n o c l o n a l antibodies to B cells Ian F. C. McKenzie and Heddy Zola Monoclonal antibodies ( Mab) have deJined fimclional T lymphoo, te subsets in mouse and man: L y - 1 / O K T d positive helper T cells, Ly- 19,,]/OKT8 positive kdler T (ells and Ly-2d/OKT8 positive suppressor T cells. An equi~,alent array q/-use~d reagents for B-cell subsets is nol ),et available - allhough many laboratorie.~ are trying to produce lhem. ht this" review lan Mekenzze and Heddy Zola summarize the Mab which are m,adable and which detecl polrmorpbic or monomorphtc determinanls on the B-cell surface in both man and mouse. T h e definition of functional T-cell subsets has been one of the most productive a n d useful exercises of the past decade. Progress in defining B-cell s u b p o p u l a tions has been slower but m a y be q u i c k e n e d by the use of M a b . We i n t e n d to review the success M a b have received so far but will exclude antibodies to surface (s) a n d cytoplasmic (c) i m m u n o g l o b u l i n (Ig), a n d Fc or c o m p l e m e n t receptors, a l t h o u g h it is clear that Bcelt subsets can now be defined in the mouse on the basis of the relative a n d absolute a m o u n t s of I g M and IgD present (Herzenberg, L. A. - personal comm u n i c a t i o n ) . M a n y of the M a b we will describe do not react exclusively with B cells - b u t this does not deny their usefulness, in the same way that a n t i - T h y - 1 anti-' bodies, not entirely T-cell specific, have b e e n powerful in the detection of T cells in the mouse. T h e p a t h w a y ( s ) taken by B cells as they differentiate from pluri-potential stem cells t h r o u g h i n t e r m e d i a t e stages to the t e r m i n a l p l a s m a cell stage has been m a p p e d in some detail, using i m m u n o g l o b u l i n expression as the principal marker, a n d since i m m u n o g l o b u l i n is the principal functional product of B cells, this is appropriate. T h e differentiation has been considered as m a t u r a t i o n along a single p a t h w a y a n d evidence for the differentiation of B cells into divergent pathways, differing in function ( c o m p a r a b l e to the T 4 a n d T8 p a t h w a y s of the T cells) is not compelling. However, there m a y be considerable heterogeneity w i t h i n the c u r r e n t l y defined m a t u r a t i o n stages, a n d M a b should help to i n t r o d u c e m o r e d e t a i l into the ' m a p ' , p a r t i c u l a r l y as they can be used to purify subp o p u l a t i o n s for functional analysis, e n a b l i n g a definition relating p h e n o t y p e to function, as has been done for T cells. Monoclonal
a n t i b o d i e s to m u r i n e B c e l l a n t i g e n s
la antigens. A large n u m b e r of a n t i - I a M a b have been described ~ 4 ( T a b l e I) a n d several s u m m a r y statements can be made: (i) M a b recognize existing specificities (la.2, 3, 7, 8, 9, 15, 20, etc.) or new specificities; (ii) the M a b confirm a n d extend the existing knowledge of la defined by alloantisera, i.e., they detect 1-A or I-E encoded specificities; (iii) one Department of Pathology, University of Melbourne, Parkville, Victoria, Australia; and Department of Clinical Immunology, Flinders Medical Centre, Bedford Park, South Australia. e Elscxwr Blotnedl~M Pres, 1983 0167-4tH0/83/[11)1)0~001~/$1
(In
interesting a n t i b o d y ~ (CE 197) detects a cross reaction b e t w e e n I-A;, a n d l-Eh- subregions; (iv) several M a b ~',= to the ' h y b r i d ' Ia.22 a n t i g e n have been described (this a n t i g e n is present in strains of H-2 haplotype k, absent in b a n d d, b u t present in ((b x d)F I mice); (v) on the basis of sensitivity to pronase a n d resistance to n e u r a m i n i d a s e a n d glycosidases, (protein - p) or the reciprocal ( c a r b o h y d r a t e - c) M a b detect two different classes of Ia molecules s lap a n d laC; e.g. several antiIa.2 or Ia.17 M a b a p p e a r to recognize the same specificity, yet one detects laP, the other IaC; (vi) the rat a n t i - I a a n t i b o d y M R C O X 6 a p p e a r s to react exclusively with the I-A [3 c h a i n '~ suggesting that the 13 chain is more i m p o r t a n t in p o l y m o r p h i s m t h a n the a chain. While of great interest in studies of i m m u n e function, a n t i - l a sera are of limited value in disc r i m i n a t i n g b e t w e e n B-cell subsets in the mouse as they are present on all or most B cells, but a b s e n t from stem cells a n d the early stages of B-cell differentiation 1°. TABLE I. Monoclonal anti-murine B-cell antibodies Designation
Reaction
Reference
A. Ia
I-A (la. 2,3,7, 8, 9, 15, stem ,clg ,slg ,PFC + 20 etc.) I-E (Ia. 7, 22) I-A and I-E (CE197) la. 22 ('hybrid') la t,, la, (la. 2, 17) MRC OX c' ([3chain)
1-4 1-4 5 6, 7 8 9
B. B-cell specific
Lyb-2.1, 2.3 Ly-m20.2 (Ly-17.2) PC-2
stem , clg +, sIg +, PFCstem?, early BY, slg +, PFC ~ B cells , PFC'
11-15 16 18
C. Rat antl-mouse
DNL 1.9, RA3-2C2, 14.Stem , cIg ÷, slg +, PFC ~ 19B5 (ATA) stem , clg +, sIg +, PFC?
10, 19, 20 26, 27
D. Potentially useful B-cell + T-cell Mab
ThB Qa-m2 LY-ml8 Ly-m19 J1 ld
(50% thymus, all B) (10% thymus, 100% T, 15%B) (15% thymus, 90% T, 80% B) (90% thymus, 50% T, 50% B) (90% thymus, 0% T, most B)
22 23 24 25 28
Immunology To&O',v,l. d, .#b. I, I(.]8.3 L~cb-2.1. The Lyb-2 locus was originally defined by conventional antisera ~t but more recently ~2 by M a b to the Lyb-2.1- specifities2.L Lyb-2 is situated on chromosome 4 and in contrast to other l:y loci has. three alleles. The M a b react with a molecule of mol. wt 45,000 which is present only on B cells, and on their Ig- precursors, but not on plasma cells. It has been suggested that the Lyb-2 site on B cells may be involved in the generation of plaque forming cells (PFC). t3-1s, Ly-m20.2. This antigen has recently been described by a series of cytotoxic M a b (Ref. 16) produced as A anti-70Z/3 or (A x C 3 H / A n ) F I anti-70Z/3 (70Z/3 is a pre-B cell tumor). Ly-m20.2 is present on B cells in spleen, lymph node; on 50% of bone marrow cells, but is not on thymus cells. Ly-m20.2 is p r o b a b l y present on all B cells as the bone marrow reaction is high and I g M - P F C were positive. The Ly-m20 locus is linked to Mls on chromosome is probably the same as Ly-17 iT, and could be related to the Mls or Ly-M loci. Like many of the murine Ly specificities which are not lymphocyte specific, Ly-m20.2 is found on liver and kidney.
PC.2. The plasma cell PC.1 alloantigenic system was defined some years ago by conventional alloantisera, and in attempts to make M a b , the PC.2 locus was described TM after a fusion of the spleen cells of ( D B A / 2 x C 5 7 B L / 6 ) F I mice immunized with the M O P C 70A plasmacytoma. PC.2 is found on several plasmacytomas and also appears to be present on both IgM and IgG PFC of all strains. No M a b to PC. 1 have been described. T h e M a b d e s c r i b e d above were m a d e after mouse/mouse immunizations and fusions and with the exception of PC.2, recognize polymorphic antigens. The next series are xenogeneic (rat anti-mouse) immunizations and recognize non-polymorphic or monomorphic determinants.
D N L 1.9. A M a b was produced i,~ by immunizing rats with 70Z/2 and detects an antigen present on all cells of the B-cell but not T-cell lineage, including sIg , cIg + cells, and both IgM and IgG PFC. In addition, a proportion of s I g - and c I g - cells were D N L 1.9 +. This is an interesting antibody as spleen colony-forming units (CFU-S) and culture colony-forming units (CFU-C) are D N L 1.9 , and with the finding of I g DNL 1.9 + cells an early B-cell precursor may have been defined.
RA
~-2C2. A n o t h e r M a b was p r o d u c e d 2° by immunizing rats with the Abelson murine leukemia virus-induced pre-B cell line R A W 112. The antibody is similar in reaction to D N L 1.9 and both M a b could recognize the same determinant. RA 3-2C2 +, I g - cells can give rise to slg + cells in vitro within 2 days, thus confirming the detection of early B cells by these two antibodies. It has also been demonstrated that a proportion of the R A 3-2C2 + I g - cells in spleen and lymph node are also Ly-2 + (S. Morse - personal communication). A similar antigen to these could be B2A2
11 which is absent from C F U - S but present on most B cells including the ' w e - p r o g e n i t o r cells '21.
14.8. Another series of rat M a b anti-murine pre-B cell tumors have been described and it is likely that the previous antibody and some of these M a b are similar. Of interest is the finding that several of this series (177.17 and 83.4) react with h u m a n cells. In addition, the 14.8 M a b reacts with a glycoprotein of mol. wt 220,000 which is the B-cell equivalent of the T-200 molecule and has also been defined in man. Extensive studies on the time of a p p e a r a n c e of antigens indicate that 19B5 (see below) appears before 14.8 and Lyb-2 antigens, both of which a p p e a r before Ia antigens in the mouse. Another series of M a b detect antigens on both T and B cells but could be useful in studying purified Bcell subpopulations. These are: ThB. The ThB locus was defined 22 by a M a b produced after a fusion using a rat spleen immunized with MOPC-104E. T h e antigen is present on all B cells, not on T cells and on 50% of thymus cells. O f interest is the finding that strains are high or low with regard to expression of ThB on B cells and the gene controlling the phenomenon is linked to Ly-6. W h e t h e r ThyB is present on early or late B cells (PFC) has not been reported. Qa-m2. The Q a series of loci (Qal-5) map to the right of the H-2 complex and are found on all T cells and a p r o p o r t i o n of B cells. T h e Q a - m 2 m o n o c l o n a l antibody recognizes a polymorphic determinant (M F 39,000 and [32M) found on all T cells, < 10% of thymus cells and only 10-15% of B cells > . It is not clear at present whether early B cells are Q a - m 2 +, but lgm PFC are Qa-m2- and IgG PFC are Q a - m 2 +. In purified B-cell populations this M a b may be useful in differentiation of the stages in B cell development. L y - m l S , L y - m l g . These antigens have recently been described 24.> and could be of interest as they react with 50% and 90% of Ia + B cells, respectively. The L y - m l 8 M a b was made against spleen cells and Ly-m 19 against the 70Z/3 tumor.
A TA (Abeison - target associated antigen). The 19B5 M a b was made by immunizing rats with mouse brain 2~', and reacts with thymus (98%), bone marrow (35%), spleen (34% - where ~17% of both T and B cells react) but not with lymph node cells. In bone marrow, the C F U - S are negative but the target cell for Abelson virus infection - presumably an early B cell is eliminated. In addition, several pre-B cell tumors are positive (e.g. 70Z/3), as are B cells in fetal liver. The B-cell regenerative capacity of bone marrow cells is impaired after treatment with the 19B5 M a b (Ref. 27).
J l l d . The J1 ld M a b reacts with red cells, thymus (not mature T cells) and with most B cells 2~, although it can distinguish B-cell subsets because primary IgM responses but not secondary IgG responses can be ablated. T h e M a b d e s c r i b e d thus p a r t l y d i s c r i m i n a t e
12
hnmunologv Today, vol. t, No. 1, 1983
between the different stages of B-cell differentiation b u t most are present on all or most B cells. However, the PC.2 a n t i g e n is confined to plasma cells, Lyb-2 is absent from these cells a n d the D N L 1.9, RA3-2C2 a n d 14.8 a n t i b o d i e s recognize the precursor of the c I g sIg- B cell. T h e a p p r o a c h for the p r o d u c t i o n of B-cell M a b m a d e so far has been to either i m m u n i z e with whole cells from spleen etc., or with B cells a n d hope for the best, or to use t u m o r s such as 7 0 Z / 3 which are regarded as ' p r e - B ' cells a n d to test on these t u m o r s or a panel of t u m o r s 29,3°used to classify B-cell differentiation steps. U n f o r t u n a t e l y , m a n y of these cells also carry m a r k e r s which represent later stages of B-cell development.
TABLE It. Monoclonal antibodies reacting with human B lymphocytes.
Monoclonal
B.
a n t i b o d i e s to h u m a n B c e l l s
Designation Reaction A.
Antibodies against 'Ia-like' molecules
Many described, including:DA2 la (p28, 33) 2.06 la (p28, 34) SI.9; S1.5 Ia (p28, 33) FMC 4 Ia (p28, 33) L203; L227 la (p28, 34) SG157 and I-E (p29, 34) and l-A (p30, 33), SGI71 respectively FMC 2 HLA-DRw4 Genox 3.53 HLA-DR-I, 2, 6 (supertypic) MC 26 IR-new
FMC 1 FMC 7 BI (p30) B2 (p 140)
Ia ( H L 4 - D R ) . A large n u m b e r of M a b reacting with m e m b r a n e 'Ia-like' a n t i g e n s have b e e n p r o d u c e d (Table II) a n d although analysis of the specific gene products detected by different M a b is incomplete, a n u m b e r of interesting features are emerging. Most of the M a b described react with n o n - p o l y m o r p h i c determ i n a n t s , but some react with H L A / D R associated polymorphic antigens. A n example is F M C 2 ~7 which reacted with some, but not all H L A - D R w 4 donors. Another, MC-26, defines a new D R specificity u n r e l a t e d to a n y k n o w n specificity TM. M o r e c o m m o n l y , p o l y m o r p h i c d e t e r m i n a n t s d e t e c t e d have b e e n ' s u p e r t y p i c ' , e.g. Genox 3.533~, which reacts with H L A - D R w 1, 2 a n d 6. T h e antibodies against n o n - p o l y m o r p h i c d e t e r m i n a n t s show considerable heterogeneity, a n d different M a b a p p e a r to detect several different gene products. For e x a m p l e L203 a n d L227 detect two different p o p u l a t i o n s of p28,32 molecules on the cell line Raji >. Such M a b will e n a b l e the identification of gene products equivalent to the m u r i n e 1-,4 a n d I - E subregions. M a n y of the antibodies described thus far p r o b a b l y identify I-E-like antigens, whilst antibodies SG 157 a n d SG 17 ! identify I-E-like a n d l-A-like molecules, respectively 36. A further d i m e n s i o n in the heterogeneity of gene products of the I region is the description of carbohydrate-defined as well as polypeptide-defined polym o r p h i s m s similar to that found in the mouse (Betts, R. L. a n d M c K e n z i e , 1. F. C. - m a n u s c r i p t in p r e p a r a tion). As only one or two M a b have b e e n described thus far which define k n o w n D R specificities, it is unlikely that equivalent antisera to the h u m a n antiD R sera will be easily produced, but the value of the existing M a b for tissue t y p i n g has yet to be determined.
Y29.55
M a t u r e B cells. M a b s reacting with B cells in blood or secondary l y m p h o i d tissue have been described (b, T a b l e II) a n d have been of some diagnostic value. For example, F M C 1 has been used in e n u m e r a t i n g ' B cells in the diagnostic l a b o r a t o r y for 2 years 3'~, a n d generally parallels sIg as a B-cell marker, whereas
Tul Ab89 H76
37 31 38
All B cells in blood 39 B cell subpopulation 40 All B cells in blood 42 Weak reaction with blood B cells but 43 strong reaction with tissue B cells Tissue B cells and B leukaemias/ 44 lymphomas, but only small proportion of normal blood B cells Sub-population of tissue B cells 45 No reaction detected with normal B cells 46 Murine antigen cross-reactive with 47 human, probably MHC product
Antibodies which are not restricted to the B-cell lineage but may provide a useful classification within the B lineage.
F8-1 l- 13 (p215) PI 153/3 BA/1 FMC 3 FMC 8 VIC-Y1 D.
31 32 33 34 35 36
Antibodies reacting only with B cells (in haemopoietic tissue)
A n u m b e r of M a b which react with h u m a n B cells have been described ( T a b l e II) which includes antibodies which are not B-cell specific b u t nevertheless show selective reactivity w i t h i n the B-cell lineage.
C.
Reference
B-cell-associated determinant of 48 leucocyte-common antigen. Strong reaction with B cells, weak with T cells. Neuroblastoma, B cells, non T-non 49 BALL. B cells, pre-B ALL, granulocytes 50 Some B cells, only some B-CLL. Most 51 T cells, monocytes. Some B cells, only some B-CLL; 52 Platelets, monocytes, granulocytes. Cytoplasmic marker on B cells and 53 monocytes.
Antibodies reacting with possible precursors of B lymphocytes
J5 (gp95) c-ALL, some normal marrow cells. VIL-A1 c-ALL, some normal marrow cells. BA/2 (p24) c-ALL, some normal marrow cells.
54 55 57
F M C 7 reacts with a B-cell s u b p o p u l a t i o n 4" a n d is useful in distinguishing p r o l y m p h o c y t i c leukaemia from chronic lymphocytic l e u k a e m i a 41. M a b B1 a n d B2 show a n interesting difference in their reactivity with blood B cells a n d B cells in l y m p h nodes 42,43. M a b Y29.55 is absent from most blood B cells, b u t is expressed on tissue B cells a n d on the m a l i g n a n t cells of B leukaemias a n d l y m p h o m a s 44. T u l shows a n even more restricted distribution, being found almost exclusively on germinal centre B cells a n d some B-cell m a l i g n a n c i e s 4~. Ab89 reacts with a limited range of Bl y m p h o m a s a n d no reactivity with n o r m a l B cells has been detected 46. M a b H76 has not been studied extensively in m a n ; it is a n a n t i b o d y p r o d u c e d against mouse B cells which cross-reacts with a n H L A - l i n k e d a n t i g e n on h u m a n B cells, possibly H L A - D R or a related p r o d u c t 47.
Immunology Today, vol. -f, No. 1, 19,~¢3
13
A n t i b o d i e s reacting w i t h B and n o n - B cells. T h e antibodies listed in Table IIc are not specific for B cells, but nevertheless provide interesting information on the classification and maturation of B cells. M a b F8-11-13 reacts with a p215 antigen which is the leucocyte-common antigen detected by other M a b and it also reacts strongly with B cells, but weakly or not at all with other leucocytes, indicating that it reacts with a B-cell associated determinant found on some molecules of the leucocyte-common a n t i g e n 4s. This relationship of F8-11-13 to the leucocytecommon antigen is of interest and suggests that differentiation may be accompanied by changes in limited parts of molecules common to m a n y related cells a striking distinction the concept of differentiation markers as from whole molecules, their presence depending on the differentiation pathway taken by the cell. PI 153/3, a M a b p r o d u c e d a g a i n s t neuroblastoma cells, also stains B cells a n d pre-B leukaemia cells 49. The BA/1 Nlab reacts with B cells, acute lymphoblastic leukaemia (ALL) cells of pre-B type, but also reacts with granulocytes~L F M C 3 reacts with some B and T cells and with chronic lymphocytic leukaemia (CLL) cells in only a minority of cases, providing information on the heterogeneity found in this disease <. F M C 8 similarly distinguishes between B-cell subpopulations, but also reacts with platelets, monocytes and granulocytes s2. Vic-Y1 is a cytoplasmic marker found in B cells and monocytes, and serves as a reminder that specific markers are not
to be found exclusively at the cell membrane~L B cell precursors. In addition to the foregoing, some important M a b react not with mature B cells, but with their precursors (Table II-d). J5 and Vii-A1 Mab react with C A L L A antigen, previously defined by rabbit antisera ~4`s'. (This marker is present on ALL cells of the ' c o m m o n ' type, c-ALL; it is also present on a small n u m b e r of cells in normal marrow, but is nevertheless very useful in the diagnosis of leukaemia). M a b BA/2 reacts with a marker which has a similar but not identical expression 57. M a b a n d B cell m a t u r a t i o n In T a b l e I I I a simplified B-cell maturation scheme is used as a framework on which to compare the monoclonal antibodies. Cells differentiating from the progenitor stern cell are considered to be committed to the B lineage when rearrangements of immunoglobulin (Ig) genes have been initiated, since these are characteristic of the B lineage. Although there is no simple test to identify such committed progenitors, pre-B cells can be readily identified by the expression of low levels of cytoplasmic immunoglobulin (cIg). Pre-B cells mature into B cells, which have detectable surface m e m b r a n e i m m u n o g l o b u l i n (slg) but no readily-detectable cIg. These sIg + cells are the B lymphocytes of the blood and secondary lymphoid tissues and are heterogeneous in terms of amount and class of slg. Finally B cells can be stimulated to
TABLE III. B-cellmaturation a Stem c e l l A.
B.
C.
pre-B
early B
late B
Plasma cell
clg +
slg +
slg ++
clg "4-+ secreted Ig
Immnnoglobulin characteristics Germ-line genes
lg genes re-arranged
Occurrence in normal blood/tissue Bone marrow
Bone marrow
Bone m a r r o w
CommonALL
pre B-ALL
Blood/secondary lymphoidorgans
Germinal centres
Leukaemia model
Null leukaemia
D.
B-committed Progenitor
CLL
PL; HCLa Muhiple myeloma Waldenstrom's macroglobulaemia
Monoclonal antibody reactivity
Anti la (p28, 33) PI 153/3 FMC 8 (p24) B1 B2 BAI BA2 (p24) J5 (gp 100) VIL-A1 FMC 1 FMC 7 FMC 3 apLL and HCL: prolymphocytic and hairy cell leukaemia; data drawn from references in Table II, 41, 59-61, and Zola el al (unpublished). Dotted lines ...... areas of particular uncertainty; . . . . . reaction with a proportion of cells; - a definitereaction.
Immunology Today, vol..'I, .¥a. 1, 1983
14
differentiate into plasma cells, the functional end cells of the B lineage. In relating phenotype to position in the differentiation pathway normal cells (b, Table III), leukaemic cells (c, Table III) or lymphoblastoid cell lines may be used, although there are considerable uncertainties in relating a leukaemic cell or cell line type to a position in the normal maturation pathway. In particular, the use of cALL + c l g - leukaemias as representatives of the B-committed progenitor is based on the observation s" that Ig gene rearrangements can be detected in cALL but not in T.ALL. The data in Table III reinforce the idea that cALL + cells are early members of the B lineage. A preliminary assignment of the reactivities of many of the Mab is shown (Table IV-d). Bearing in mind the heterogeneity of the data (obtained from studies of leukaemic cells, cell lines, and on normal cells) the assignment must be tentative. The forthcoming International Workshop on H u m a n Leucocyte Differentiation Antigens should clarify the reactivities of these antibodies. It will be noted that several Mab detect antigens present on most of the B lineage, but none encompass the entire maturation sequence ([3-2 microglobulin and HLA do appear throughout the lineage, and on most other nucleated cells). One antigen (detected by F M C 3) seems unrelated to maturation, being found on a proportion of cells at all stages. Several of the antibodies listed show a different reactivity with B cells of lymphoid organs and B cells in blood. Thus, T u l and Y29-55 are absent from normal circulating B cells but present on tissue B cells and circulating leukaemic B cells. B2 reacts strongly with sessile B cells in lymphoid tissue but weakly with circulating B cells. Furthermore, maturation from primary to secondary follicles is accompanied by changes in the relative expression of B1 and B2 (Ref. 59). None of the antibodies described so far is restricted to stem cells or the B-committed progenitor. This probably reflects the screening panels that have been used to detect Mab, and the use of new tumors, screening on a panel of tumors or performing functional screening may provide the answer. For example, M. Beveridge (personal communication) is assaying by the CFU-S technique - keeping the hybrid colonies growing for 7 days until the results are available. In addition, it will be necessary to detect reactivity with extremely small (<1-5%) proportions of bone marrow cells because precursors may only b e present in such small numbers (note: none of the markers which define T-cell subsets in the mouse are present on <10% of cells.) Clearly more Mab are needed and given these, our understanding of B-cell differentiation and function should increase greatly o v e r the next few y e a r s , Acknowledgment The studies reported herein were supported by funds obtained from the National Health and Medical Research Council of Australia.
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