Eur. J. Immunol. 1990.20: 771-777 Luc Reiniiger, Takanori Shibata, Shoichi Ozaki+*, Toshikazu Shirai+, Jean-Claude JatonO and Shozo Izui Departments of Pathology and Medical Biochemistryo, University of Geneva, Geneva and Department of Pathology+, Juntendo University School of Medicine, Tokyo

V region sequences of pathogenic anti-MRBC autoantibodies

Variable region sequences of pathogenic anti-mouse red blood cell autoantibodies from autoimmune NZB mice* New Zealand Black (NZB) mice spontaneously develop a severe autoimmune hemolytic anemia due to the production of anti-mouse red blood cell (MRBC) autoantibodies. The contribution of variable region genes and somatic mutations in the pathogenicity of anti-MRBC autoantibodies was investigated by mRNA sequencing of eight NZB anti-MRBC monoclonal autoantibodies, among which five are capable of inducing anemia in BALB/c mice. Here we report that at least threeVHgene families (5558,5606 and 3609) and fiveV, subgroups (V,8, 9,19,21 and 28), in combination with several D, JH and J, gene segments, encode anti-MRBC autoantibodies.Thus, the NZB anti-MRBC autoantibodies, whether pathogenic or not, are encoded by a large number of immunoglobulin gene elements and by members of known VH and V, gene families with preferential usage of VH gene families most distal to the D regions. The presence of several mutations in the JHgene segments of both IgM and IgG anti-MRBC autoantibodies, whether pathogenic or not, strongly suggests that their VH regions may be highly mutated and that the mechanism of somatic diversification might be important in the generation of anti-MRBC autoantibodies. Our results support the idea that anti-MRBC autoimmune responses are likely to be generated by an antigen-driven mechanism.

1 Introduction Antibody diversity is generated in part by the combinational assembly of VH-D-JHand VL-JLgene segments, and by possible extensive somatic diversification of V region gene segments which provides an almost unlimited potential for V region diversity [l] and hence autoantibody specificity. Previous sequence studies on various autoantibodies have demonstrated that IgM autoantibodies such as anti-DNA, anti-IgG rheumatoid factors (RF) or anti-bromelaintreated mouse red blood cell (BrMRBC) autoantibodies are encoded by germ-line genes [2-71, and that considerable somatic mutations in the complementarity determining regions (CDR) can be found in IgG anti-DNA autoantibodies and IgG RF [8, 91. Although it is now clear that Ig germ-line genes encode a variety of autoantibodies and that somatic mutations may well occur in such antibodies during the course of autoimmune diseases, the pathogenic activity of autoantibodies so far analyzed has never been directly demonstrated in vivo.Therefore, it is important to define the genetic origin of pathogenic autoantibodies and the contribution of somatic events to the pathogenic activity of autoantibodies. [I 81791

*

771

This work was supported by grants no. 31-9217.87 and 3126461.89 from the Swiss National Foundation for Scientific Research, by a grant from the Ministry of Education, Science and Culture, Japan, by the Swiss Confederation acting on the proposal of the “Commissions Fkdkrales des Maladies Rhumatismales”, and by the Roche Research Foundation. Present address: Laboratory of Immunology, Department of Clinical Research, Utano National Hospital, Kyoto 616,

To address this question, we have prepared eight antiMRBC mAb from unprimed anemic NZB mice. Five of them were found to be able to induce anemia in nonautoimmune mice (*, [lo]). By analyzing mRNA nucleotide sequences encoding the H and L chain V regions of these eight anti-MRBC mAb, we have unraveled the genetic elements encoding these pathogenic anti-MRBC autoantibodies, and we have attempted to determine the possible contribution of somatic mutations to their pathogenic activity. In addition, since anti-BrMRBC autoantibodies were found to be preferentially encoded by a new VH gene family, vH11, and a single V, gene family [7, 11, 121, it was of interest to determine whether anti-MRBC hemolytic autoantibodies use a restricted set(s) of genetic elements as do anti-BrMRBC autoantibodies. Our results indicate that NZB anti-MRBC autoantibodies express several different VH,V,, D, JH and J, genes with a predominant utilization of the 5’ most distal VH families.The presence of several mutations in the JH gene segments, even for IgM anti-MRBC mAb, suggests that the mechanism of somatic diversification might be important in the generation of anti-MRBC autoantibodies.

2 Materials and methods 2.1 mAb

Hybridomas used in this study were derived from the fusion of spleen cells from unprimed 6- to 10-month-old NZB A mice (obtained from the Bomholtgard, Ry, Denmark) with the myeloma cell lines NS2 or X63-Ag8.653 as described Japan. (*, [lo]). An indirect RIA was used to detect the antiMRBC autoantibody activity of the eight mAb*. Briefly, Correspondence: Shozo Izui, Department of Pathology, CMU, 1, 50p1 of a 5% MRBC suspension (in 1% BSA-PBS) rue Michel Servet, CH-1211 Geneva 4, Switzerland obtained from BALB/c mice, was incubated overnight at Abbreviation: (Br)MRBC: (Bromelain-treated) Mouse red * Shibata et al., submitted for publication. blood cell 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

0014-2980/90/04O4-0771$02.50/0

772

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Table 1. In vitro binding to MRBC and pathogenic activity of eight anti-MRBC mAb mAb

fn vitro binding’)

lElO (1.4 4C8 ( i , x ) . 103-7E ( P . x ~ ) 106-10E (p,xy) CP3 23-24 (1,x)C) 41-5C ( P , x ~ )

34.0 20.0 60.7 43.5 1.5 1.2

31-9D (YI,~) 105-2H ( y l , ~ )

31.1 29.3 24.4 27.9 2.7

343c (Y2a,X) 34-28 ( Y ~ ~ . x ) C ) Hy-1.2 ( Y Z ~ . X ) E )

Hematocrit (%) 3 days after mAb injectionh) 12.5 pg SO pg 200 pg 5wPg

40 42 ND ND ND ND 28 ND 45 ND ND

29 33 47 47 ND ND 17

45 34 43 ND

Kill miced) Kill mice 48 46

45 47 in 43 13 46 45

4 “Cwith 100 pl of hybridoma culture SN containing 1-2 pg of each mAb. After washing three times with PBS, the MRBC were incubated overnight at 4°C with 100 p1 of 1251-labeledrabbit anti-mouse IgG (Dakopatts, Copenhagen, Denmark) or 1251-labeledrat anti-mouse p chain mAb, LO-MM-9 [13]. After three washing steps, the radioactivity bound to MRBC was counted.To determine the pathogenic activity of the anti-MRBC mAb, hematocrits were measured daily after a single i.p. injection of hybridoma culture SN, or ascites fluids into BALBk mice. IgM anti-BrMRBC mAb (CP323-24; [14]) IgM (41-5C) derived from NZB mice and IgG2, anti-DNP mAb (Hy1.2; [15]) were used as negative controls. Details of in vitro and in vivo activities will be described elsewhere*.

ND ND 48 47 46 48 Kill mice 30 Kill mice 45 46

a) Anti-MRBC activities of IgM and IgG mAb are represented as the percentage of lZ5I-rat anti-mouse p chain mAb or of lEI-rabbit anti-mouse IgG bound to intact MRBC, respectively. Hy-1.2, control IgGza mAb [15]; CP323-24, IgM anti-BrMRBC mAb [14]; 41-5C, control IgM mAb. b) BALBk mice received a single i.p. injection of different amounts of mAb. Mean hematocrit value of three to five mice. c) Injection of 5 mg of mAb induced no anemia in recipient mice. d) The recipient mice died of acute anemia 2-3 days after the mAb injection.

5’d(GGACCATCACTGTAAGG) (VH34-2B; 52a-57), 5‘d(CCClTTCACAGACTCAGC) ( V ~ l E 1 0 ;60-65) and 5‘d(CTGTCCAATCCACTCAAG) (vH4c8; 45-50). For L chain mRNA sequencing, a S’d(TGGATGGTGGGAAGATG) C, (116-122), a 5’d(GCCTCCACCGAACGTCC) J,1 (96-lOl), a 5’(CCCAGCACCGAACGTGA) J,5 (96-101) and the five following V, primers were used: S‘d(AACGGAATCTCCCAAC) (VX103-7E; 91-96), 5‘d(TCTACTATAATATTGCTG) (VX31-9D; 89-94), 5’d(AA’rr?.CCTI’ACCITGCTG) (Vx34-3C; 89-94), S’d(GCCACTAAACCTGGCTGGG) (VX1E10; 58-64), and 5’d(CACTGCCTGTGAAGCG) (VX4C8;61-66).

3 Results 2.2 Nucleotide sequencing

This procedure was carried out as described previously [7]. Briefly, poly(A)+ RNA were extracted from hybridomas grown in tissue culture or from fresh tumors according to the urea-LiC1method [161, and were purified by oligo(dT)cellulose column chromatography [17].The H- and L chainencoding fractions of mRNA were separated on sucrose gradients [181. mRNA nucleotide sequencing was carried out according to the method of Hamlyn et al. [19]. Each sequence was detrmined by using ~ ~ ~ ~ S - r a d i o l a bdATP, eled dCTP and dGTP (600Ci/mmol = 22.2 TBq/mmol, Amersham Int., Amersham, GB).

3.1 Pathogenicity of anti-MRBC mAb from NZB mice

Eight hybridomas producing IgM (103-7E, 106-10E, lElO and 4C8), IgGl (105-2H and 31-9D), IgG2, (34-3C) and IgG2b (34-2B) anti-MRBC mAb were obtained from four separate fusions with spleen cells of 6-10-month-old NZB mice.Their in v i m binding activities towards intact MRBC, as determined by an indirect RIA, is shown in Table 1. BALB/c mice which received a single i.p. injection of mAb (4C8,1E10,34-3C,31-9D or 105-2H)developed an anemia, as documented by a marked decrease in hematocrit values following the injection. As apparent from Table 1, the pathogenic activity substantially differed among the mAb. In contrast, mAb 34-2B (IgGZb), 103-7E(IgM) and 106-10E (IgM) were not able to induce an anemia even after the injection of 5 mg of mAb. As a control, none of the 2.3 Oligonucleotide primers antibodies IgM (41-5C), IgM anti-BrMRBC (CP3 23-24; [14]) and IgG2, anti-DNP (Hy-1.2 [15]) caused anemia in Synthetic oligonucleotides synthesized with a System 200A BALBk mice, under the same conditions. Beckman DNA synthesizer (Beckman Instruments, Palo Alto, CA) were used as primers. For H chain mRNA sequencing, a 5’d(GCT(=TCGCAGGAGAC) C,1 primer 3.2 Molecular basis of the VH regions of anti-MRBC complementary to C,1 codons 125 to 129 (125-129), a mAb 5’d(GGCCAGTGGATAGAC) C, (121-125) primer, and the six following VH primers were used: 5’d(CAATC- The nucleotide sequences of the portions of mRNA CACTCAAGG) (V~103-7E, 106-10E, 34-3C; 44-49), encoding VHregions and the corresponding deduced amino S’d(AGTACTAGTACTACCAGG) (V~105-2H;52a-57), acid sequences of the eight anti-MRBC mAb are presented 5’d(GCCACTCCAGACCCTTCCC) (V~31-9D; 42-48), in Fig. 1. The V region gene characteristics of these anti-MRBC mAb are summarized in Table 2. The comparison of the VH sequences with members of the eleven * Shibata et al., submitted for publication.

V region sequences of pathogenic anti-MRBC autoantibodies

Eur. J. Immunol. 1990.20: 771-777

773

106-1M 103-7E 105-211 34-3c 34-28 31-90 1ElD 4ca

106-1DE 103-7E 105-21

34-y 35-20 31-$9 (El0 4ca

I

M

1ElD 4ca

106-1DE 103-7E 105.21 34-3c 34-28 31-90 lElO 4C8

a b c

90

I 1Wh i j

I

-1 k

110

Figure 1. Nucleotide sequences of mRNA encoding the VH regions of eight NZB antiMRBC mAb and predicted amino acid sequences. N (X) undetermined base (residue); dots above a base (residue) indicate the probable base (residue) at that position. Numbering of amino acid residues (one-letter code) and CDR is according to Kabat et al. [23].

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L. Reininger,T. Shibata, S. Ozaki et al.

774

nucleotide substitutions. The nucleotide substitutions are presumably not due to polymorphic variations between BALB/c and NZB JH gene segments, because these substitutions are not present at the same position in all mAb. When considering the amino acid sequences, one substitution for 4C8 mAb, two for lElO and 103-7E mAb, and three for 31-9D and 105-2HmAb are revealed,while no substitutions for 34-3C and 106-10E mAb. This finding strongly suggests that the majority of mAb, both IgM and IgG, regardless of their pathogenicity, contain several somatic mutations resulting in amino acid substitutions throughout their VH segments.

Table 2. V region gene usage in eight NZB anti-MRBC mAb

Fusion 1

2 3 4

mAb

VH

106-10E 103-7E 105-2H 34-3c 34-2B 31-9D lElO 4c8

5558 5558 5558 5558 5558 3609 5606 5558

H chain D

L chain JH

vx

Jx

2 4 4 1 3 1 3 3

21 21

1 2 1 2 1 5 2 5

N Q52

sP2.2 N sP2.5 SP2.314 FL16.2

SP2.5I718

2

8 21 9 19 21 8

3.3 Molecular basis of the VL regions of anti-MRBC mAb

Y

T

D

Y

I

Q

Q

Q

T

T

L

T

V

B

B

TAC TTT GAC TAC TGG GGC C M GGC ACC ACT CTC ACA GTC TCC TCA

JK2

--_ --T ___

106-10E

U

r

A

Y

I

___ ___ ___ ___ ___ ___ ___ ___ ___ _ _ _ Q

Q

Q

T

L

V

T

V

S

A

TGG TTT GCT TAC TGG GGC C M GGG ACT CTG GTC ACT GTC TCT GCA

Y J84

Y

A

M

D

Y

I

Q

Q

Q

T

B

V

T

V

B

B

TAC TAT GCT ATG GAC TAC TGG GGT C M GGA ACC TCA GTC ACC GTC TCC TCA

D

I

Figure2. Comparison of the JH nucleotide sequences of antiMRBC mAb to BALB/c JH germ-linegenes.Theprotein sequences in the one-letter code are indicated above the nucleotide sequences.The nucleotide sequencesfor BALB/cJH gene segments are taken from [23]. See legend to Fig. 1.

selves (103-7E, 105-2H, 106-10E, 34-2B, 34-3C and 4C8), 13.6%-24.5% nucleotide substitutions were found, suggesting that they are likely to derive from different members of the 5558 VH family. The D segments derive from DSP2.2 (105-2H), 3/4 (31-9D), 5 (34-2B), 5/7/8 (4C8), DFL16.2 (1E10) and DQ52 (103-7E) minigenes [23], with several nucleotide substitutions and additional flanking N sequences (103-7E and 34-2B; [24]), or may be entirely encoded by N segments (106-10E and 34-3C). The JH segments derive from the J H (31-9D ~ and 34-3C), J H (106-10E), J H (34-2B7 ~ 1E10 and 4C8) and J H (103-7E ~ and 105-2H) gene segments [23]. Notably, there is no obvious amino acid sequence relatedness in the CDR among these mAb. None of the VH segments reported here was found to be identical in sequence to any known germ-line VH genes. Thus, it is impossible to make a direct assessment of the incidence of somatic mutations. However, there is evidence for somatic mutations in JH segments of all eight mAb except for 34-2B mAb (Fig. 2). By comparison with the BALBk germ-line gene segments, the anti-MRBC mAb JH segments were found to contain one (106-10E, 34-3C), two (1E10, 4C8), three (103-7E, 31-9D) or five (105-2H)

Nucleotide and deduced amino acid sequences of V, regions of the eight anti-MRBC mAb are shown in Fig. 3. Their structural genetic elements are summarized in Table 2. The V, regions are quite different and can be assigned to the five following V, subgroups by considering the N-terminal amino acid sequence data through the first invariant tryptophan [25]: V,8 (4C8), V,9 (34-2B), V,19 (31-9D),VX21(103-7E, 106-10E, lElO and 34-3C) and V,28 (105-2H).When compared with the entirev, region, up to 90% to 97% nucleotide homologies were found with individual members of their corresponding V, subgroups [23], except for 34-2B which displays only 75% identity with one member of the V,9 subgroup. It should be noted that even the 103-7E, 106-10E, lElO and 34-3C mAb (V,21 subgroup) differ from each other by 10% to 16% nucleotide substitutions, suggesting that they derive from distinct germ-line genes. The 4C8 mAb carries a deletion of codon 95. There is no preferential utilization of a particular J, segment, since J,1 (105-2H, 106-10E and 34-2B), J,2 (103-7E, 34-3C and 1E10) and J,5 (31-9D and 4C8) gene segments were found [23]. Due to technical difficulties in determining the nucleotide sequences of the J, segments of several mAb, it is not possible to know whether the J, segments used by the anti-MRBC mAb differ from their corresponding germ-line genes. As with theVH segments of these antibodies, V, CDR differ considerably among the eight mAb.

4 Discussion The present work reports the first molecular analysis of autoantibodies whose pathogenicity has been directly ~demonstrated in vivo. The analysis of mRNA sequences encoding the VH and VL regions of five pathogenic and three non-pathogenic anti-MRBC mAb from NZB mice reveals several important points: (a) the pathogenic antiMRBC autoantibodies are encoded by VH5558,3609,5606 and V,8, 19, 21 and 28 genes, indicating that their pathogenicity is not determined by a paticular set of V region genes; (b) the anti-MRBC Ig V region repertoire arises from the genetic repertoire that encodes antibodies directed against exogenous antigens, but is not related at all to that of anti-BrMRBC autoantibodies; (c) there is no restriction for utilization of the VH gene families most proximal to the D region locus, but rather an overrepresentation of the most distal VH gene families and (d) there exists a high incidence of somatic mutations within the JH

Eur. J. Immunol. 1990.20: 771-777

V region sequences of pathogenic anti-MRBC autoantibodies

gene segments, even for the IgM autoantibodies and independently of their pathogenic activity, suggesting that the mechanism of somatic diversification might be important in the generation of anti-MRBC autoantibodies. 10

20

The nucleotide sequence analysis of the eight anti-MRBC mAb indicates the involvement of many different VH,V,, JH, J, and D gene segments in the generation of anti-MRBC autoantibodies in the NZB mouse. The lack of any

I

an1

30

a b c d . 1

....

106- WE

W U T ~ G I G C l W C C C U T C T C U G l l C l T T ~ I C l C T C l C l A ~ ~ ~ T A l C C l ~ ~ I ~ T C l l. TWT TlWACCMll A.C l l l T

Im-7E

.........T..A.A..G.....T.....C..A....lA..G..c...n.....

. .

.c.C....ul........c...~.c.c...

31-90

..C......T..A....W....yyU-..C..CT..~~..~....~.C....~~......~ .....-......c.c.................. ............A........................T.. ......................c..............C.............A........A..G..T....... .....C-A-A ...i c..~.........*G.c..l...u..c..c ..~.c.~~~.....~.~...c.~......c.~...........................A.A..C.A-.lA.l.uU.*G.c.cC. .Acl.*GCc..

(El0

.........n............---..n

l(K-2Y

34-x 34-28

.........A..T.*..G......

106-llx 103-7E

lM-ZH

34.x 34-28 31-90 lElD

4a

106-lo€ 1OS-7E 105-2M

34-x

36-28 31-PD 1ElD

4a 106-la 105-7E lpn-tw

34-x

36-2n 31-90 1ElO

4a

106-1a 103-7E tm-2n 34-x

34-28 31-90 1ElO

bB

.....---yT--......N..........T

775

..

l...... A.

Ac..Ic..c.A.

lif...~.. OG &+.TG----C-a.

A.

......W..cTG......

A.

T-C--CC-~A---~---*G-~--*G------TT--~--~-----~T.----C-G--C~--TT~~~-~~~~-C-~--~L-~C-~C

776

Eur. J. Immunol. 1990. 20: 771-777

L. Reininger,T. Shibata, S. Ozaki et al.

correlation between pathogenic anti-MRBC mAb and V any known germ-line gene. Therefore, a direct estimation region gene usage strengthens the idea that the develop- of the frequency of somatic mutations in anti-MRBC VH ment of the autoimmune hemolytic anemia in the NZB and VL gene is not possible. Because the D segments are mouse is not determined by a primary defect of the Ig V frequently subjected to alterations occurring during VHregion gene repertoire, in view of essentially identical D-JH rearrangements, they cannot be used to analyze the restriction fragment length polymorphism patterns in non- mutation rate. However, the presence of several mutations in the JH gene segments of both IgM and IgG anti-MRBC autoimmune and autoimmune-prone mice [2, 3, 26-28]. Furthermore, the fact that all VH and VL rearranged gene autoantibodies suggests that their VH regions could likesegments could be assigned to knownV region gene families wise contain numerous somatic mutations. The fact that suggests that the spontaneously arising NZB anti-MRBC even IgM anti-MRBC autoantibodies are likely to be autoantibodies originate from the same repertoire as that mutated is in marked contrast with other IgM autoantibodused by antibodies elicited against foreign antigens, as ies (anti-BrMRBC, anti-DNA or anti-IgG), which do not already demonstrated for other autoantibodies [29]. In fact, exhibit apparent somatic mutations in their V region gene the contribution of several VH gene segments to the segments [2-71. Since the recent molecular analysis of diversity of anti-MRBC autoantibodies reflects the repre- human IgM anti-RBC autoantibodies has suggested that sentation of eachVHfamilies in the mouse IghV locus, since the anti-RBC reactivity is present in the germ-line reperthe largest 5558 VH family is found to encode six out of the toire [41], it is likely that the somatic mutations observed in eight anti-MRBC mAb, and the 5606 and 3609 VHfamilies, theV region genes expressed by MRBC autoreactive clones which account for two other largeVH families [20], encode reflect the selection of these autoantibodies by an antigenthe other two anti-MRBC mAb. Thus, in our small driven mechanism rather than a polyclonal B cell activacollection, pathogenic anti-MRBC autoantibodies appear tion. This is consistent with the recent demonstration of to be heterogeneous. The diversity of the MRBC antigenic preferential replacement mutations in CDR in IgG antideterminants certainly accounts for the observed hetero- IgG and anti-DNA mAb from MRL-lpdlpr mice [8,9]. In geneity of the anti-MRBC mAb, since our recent analysis this regard, it is worth noting that spontaneous anti-MRBC has shown that the eight anti-MRBC mAb recognize at autoantibody response can occur in the complete absence least three different groups of antigenic determinants*. of polyclonal B cell activation in lethally irradiated young Nevertheless, it should be noted that lElO mAb apparently NZB mice reconstituted with BM cells from old NZB mice has a specificity similar to that of 4C8, as determined by (Reininger et al., submitted for publication). reciprocal inhibition assay*,yet expresses different VH and The finding that both non-pathogenic and pathogenic V, genes. anti-MRBC mAb contain somatic mutations raises the It has been demonstrated that VH gene families localized question of the role of the mutations in the pathogenesis of most proximal to the D region locus rearrange frequently in autoantibodies. In studies of conventional anti-non-self B cells from fetal or neonatal mice as well as in pre-B cell antibodies, increased frequency of replacement mutations lines [30,31], as compared to adult mature B cells, which is associated with high-affinity antibodies [42-441. SimilarutilizeVH gene families at a frequency that correlates with ly, it may be that some somatic mutations contribute to a family size [32]. A similar biased utilization of VH gene more efficient binding to MRBC, thereby enhancing their families proximal to the D region locus by autoantibodies pathogenic activity in vivo. However, it remains to be was reported by several investigators [33-361. However, determined whether the pathogenicity of the anti-MRBC this was not confirmed by others [2, 3, 8, 9, 371. Our autoantibodies is indeed created by somatic diversification observation that all eight anti-MRBC mAb are encoded by of the germ-line repertoire. Expression of the correspondmembers of the 5’ most distal VH gene families further ing germ-line genes or site-directed mutagenesis of the argues against the preferential rearrangement of VH famil- mAb would help determine the contribution of the somatic ies proximal to the D regions for the generation of mutations in the pathogenicity of anti-MRBC autoantibodies. Certainly, the H chain Ig classes play an important role autoantibodies. in the pathogenesis of anti-MRBC autoantibodies by It is significant that there is no relation between the determining different effector functions such as compleanti-MRBC autoantibody V gene repertoire and that of ment-dependent hemolysis, FcR-mediated phagocytosis or anti-BrMRBC autoantibodies, which predominantly use a multivalency-induced hemagglutination. Further examinavH11-v,9 gene combination [7,11, 121.Therefore, distinct tion of the fine antigenic specificities and of the role of the B cell populations are likely to be involved in these two isotype of pathogenic anti-MRBC autoantibodies may help autoantibody responses. In this context, it should be elucidate the molecular mechanisms responsible for the mentioned that the anti-BrMRBC autoantibodies are development of autoimmune hemolytic anemia. selectively produced by the Ly-lf B cell population [38], Wegratefully acknowledge Dr. A . Siissman, Beckman, Geneva, for and the VHll family associated with anti-BrMRBC specif- kindly preparing oligonucleotides and Dr. A . Lussow, CMU, icity has been shown to be preferentially expressed in this Geneva, for critically reviewing the manuscript. particular B cell population [39]. At present, it is not known whether anti-MRBC autoantibodies are produced by Ly-l+ Received December 11, 1989. B cells. Although all theVH and VLgenes encoding the anti-MRBC mAb investigated could be unambiguously assigned to knownvgene families, none of them was found identical to

*

Shibata et al., submitted for publication.

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Variable region sequences of pathogenic anti-mouse red blood cell autoantibodies from autoimmune NZB mice.

New Zealand Black (NZB) mice spontaneously develop a severe autoimmune hemolytic anemia due to the production of anti-mouse red blood cell (MRBC) auto...
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