hnmunologl' Today, z,ol. l, No. l, 198d
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
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
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.
~-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
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.
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.
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.
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
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