Idiotypy of anti-Rh antibodies

Blood Transfusion and Immunohaematology Tome XXVII. -- N° 6. -- 1984

715

Idiotypy of anti-Rh antibodies b y J. de S a i n t M a r t i n a n d A. E y q u e m Laboratoire d'Immunoh6matologie et d'Immunopathologie, Institut Pasteur, PARIS.

I

NDIVIDUAL antigenic specificity of h u m a n proteins has been described by KuNKEL, MANNIK and WILLIAMS in 1963 [17]. At the same time, OUDIN and MICHEL Observed " u n e nouvelle f o r m e d'allotypie des globulines g a m m a du sdrum de lapin, a p p a r e m m e n t li6e la fonction et g la s-p6cificit6 a n t i c o r p s " [23a]. Three years later, this new p r o p e r t y of antibodies was called " I d i o t y p y " [23b]. Monoclonal or restricted heterogeneity immunoglobulins have been involved in initial studies p e r f o r m e d with h u m a n antibodies: multiple myeloma proteins [30], cold agglutinins [33], r h e u m a t o i d factors [12], monoclonal IgM f r o m Waldenstr6m's macroglobulinemia [7]. In healthy individuals, idiotypy of Rh antibodies has been studied in two laboratories as f r o m 1976 [22, 26]. Another aspect has been shown by GEHA and WEINBERGwho have p r o d u c e d and characterized anti-idiotypic (anti-id) sera to h u m a n anti-tetanus antibodies [14]. All these presented data have d e m o n s t r a t e d that anti-id sera could be p r o d u c e d with heterogenous antibodies. They have also shown cross-idiotypic specificities among antibodies f r o m non related people, specially in the Rh system [27]. Idiotype-bearing B and (or) T lymphocytes have been observed in h u m a n multiple myeloma, Waldenstr6m's macroglobulinemia and r h e u m a t o i d arthritis [25, 2, 8]. Anti-idiotypic sera have been p r e p a r e d against different autoantibodies by several workers: anti-DNA [35], anti-acetylcholine receptor [18], anti-thyroglobulin [19], anti-hepatitis B surface antigen

716

S A I N T M A R T I N (DE) et coll.

[16], IgG f r o m the cerebrospinal fluid of patients with multiple sclerosis [9]. S h a r e d idiotypic specificities have been observed in different kinds of auto-antibodies, specially in cold agglutinins [10], r h e u m a t o i d factors [5] and anti-DNA antibodies [29, 31, 35]. Furthermore, naturally occurring anti-anti-DNA have been d e m o n s t r a t e d in sera f r o m inactive systemic lupus e r y t h e m a t o s u s (SLE) patients, as well as in sera f r o m n o r m a l individuals who had contact with lupus patients; the anti-anti-DNA activity could not be detected in active S L E sera [1, 34]. During a n o r m a l i m m u n e response, auto-antiidiotypic antibodies have been shown in the s e r u m f r o m individuals who received a b o o s t e r injection with tetanus vaccine [13]. Anti-id antibodies could be involved in i m m u n e complex formation, as it has been shown in mixed cryoglobulinemia [15] and in IgA deficiency [6]. In the present study, we p r o d u c e d anti-id sera in rabbits immunized against Rh antibodies. Some of these sera were shown to be specific for the immunizing antibody. By contrast, o t h e r sera could p e r m i t detection of cross-idiotypic specificities shared b y different anti-D antibodies. F u r t h e r m o r e , we followed the evolution of idiotypic specificities of Rh antibodies f r o m one Blood Donor: LOR for a nine years period. Id-anti-id interactions have b e e n demonstrated: by means of a rosette assay, we observed r e c e p t o r s for id d e t e r m i n a n t s on some peripheral b l o o d lymphocytes (PBL), just as Rh antibodies are decreasing.

MATERIAL AND METHODS A n t i - R h sera were collected f r o m Rh negative blood donors, immunized against incompatible R h + red blood cells (RBC). Most of the antibodies contained anti-D specificity alone. A n t i - i d i o t y p i c sera were raised by means of immunization of rabbits (R) with Rh antibodies obtained b y heat elution, according to a p r o c e d u r e described elsewhere [26]. Sera were r e n d e r e d antiidiotypic specific by s e q u e n t i a l absorptions on h u m a n material: red blood cells, pooled immunoglobulins (Ig) and Fab'2 fragments covalently b o u n d to Sepharose 4B.

After suitable absorptions, sera showed n o reaction with sheep red cells coated with pooled h u m a n IgG or Fab'2 fragment. Rabbit and h u m a n IgG were isolated b y sodium sulfate precipitation, followed by gel filtration on Sephacryl S-300. Fab'2 fragments were p r e p a r e d b y pepsic digestion. F u r t h e r purification was

IDIOTYPY OF ANTI-Rh ANTIBODIES obtained by filtration protein A.

on

717

Sephadex

G-150 and

absorption

with

Agglutination and agglutination inhibition experiments. Agglutination titers of anti-idiotypic sera were d e t e r m i n e d using ORh+ (DCeee) RBC sensitized with the immunizing antibody. Inhibition tests were p e r f o r m e d by mixing equal volumes of anti-id sera and anti-Rh sera. Isolation of peripheral blood lymphocytes: L y m p h o c y t e s were collected f r o m anti-Rh Donors; they were isolated f r o m plasmapheresis bags by centrifugation on IsopaqueFicoll according to p r o c e d u r e s described by BOYUM [4]. Adsorbed Ig were r e m o v e d by incubation of the lymphocytes for five hours to + 4° C, followed by 3 washings in RPMI medium. Macrophages were excluded by iron p o w d e r ingestion. EA-Rh rosette forming cells (EA-Rh): EA-Rh rosettes were p e r f o r m e d according to p r o c e d u r e s described previously [28]. They were observed after reaction of isolated lymphocytes with O R h + RBC sensitized with anti-Rh Fab'2 fragments belonging to the same d o n o r as the lymphocytes.

RESULTS I. - -

AntMdiotypic

sera

After absorption with h u m a n RBC, Ig and Fab'2 fragments, rabbit sera strongly agglutinated O R h + RBC coated with the immunizing Rh antibodies or Fab'2 fragments (Table I). TABLE I Agglutination titers of R.605 anti-idiotypic serum to ,G~, anti-Rh antibodies. AGGLUTINATION TITER

O Rh+ RBC coated by Rabbit 605 anti-id serum

~,G,> Rh antibodies ,~G,~ Fab'2 Rh antibodies

256 256

Rabbit 605 absorbed with ,,G,, <
0

Rabbit 605 absorbed with eG,, eG,~ Rh antibodies depleted Rh antibodies serum

256

Control: Anti~Ig serum

,,G,, Rh antibodies -G,, Fab'2 Rh antibodies

i.

256 64

718

SAINT MARTIN (DE) et coll.

Inhibition of the agglutination reactions was obtained after absorption with the h o m o l o g o u s anti-Rh serum. Depletion of anti-Rh antibodies f r o m anti-Rh s e r u m was obtained b y a b s o r p t i o n s on O Rh-t- RBC. None of the agglutination reaction was inhibited b y the depleted serum. By these criteria, rabbit sera can be used for study of idiotypic specificities f r o m anti-Rh antibodies. II. - - C r o s s - i d i o t y p i c s p e c i f i c i t i e s of anti-Rh a n t i b o d i e s : agglutination reactions

The five anti-LOR sera agglutinated O R h + RBC coated with LOR antibodies. By contrast, RBC coated b y Rh antibodies f r o m 32 different donors were not agglutinated by anti-LOR sera. The seven a n t i - " G " sera agglutinated cells sensitized by " G " antibodies; some of t h e m agglutinated red cells coated by 10 out of 32 samples. Anti-MOZ and anti-BRA sera have s h o w n similar p a t t e r n s (Table H). Various agglutination titers were observed. The strongest reaction was always obtained w h e n anti-id sera reacted with RBC coated with the immunizing antibody. RBC coated with Fab'2 f r a g m e n t s were also effective for detection of cross-idiotypic specificities. No agglutination reaction was obtained using anti-C, anti-E, anti-c, anti-Fy ~ or anti-Kell coated red cells. n i . - - C r o s s - i d i o t y p i c s p e c i f i c i t i e s of anti-Rh a n t i b o d i e s : agglutination inhibition experiments

Inhibition of the cross-idiotypic systems was obtained with homologous anti-Rh sera; it was n o t observed with depleted anti-Rh serum. Attemps of inhibition of anti-idiotypic sera by different anti-Rh cross-reacting sera have been carried out: - - Decrease of h e m a g g l u t i n a t i o n titer of the h o m o l o g o u s system was not observed with cross-reacting anti-Rh sera. - - Inhibition of a given cross-idiotypic system was obtained with the c o r r e s p o n d i n g anti-Rh serum. - - Groups of cross-idiotypic systeI~IS could be a r r a n g e d on the basis of preliminary results. IV. - - A g g l u t i n a t i o n i n h i b i t i o n b y a n t i - i d i o t y p i c s e r a Anti-id sera were f o u n d to inhibit agglutination of R h + RBC by anti-Rh s e r u m ( T a b l e H I ) . Ratio of inhibition was estimated b y the

IDIOTYPY OF ANT1-Rh ANTIBODIES

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720

SAINT MARTIN

(DE) et coll.

reduction of hemagglutination titer of the anti-Rh sera. For example, after a b s o r p t i o n with antidd BRA serum, agglutination titer of BRA s e r u m d r o p p e d f r o m 1/1000 to 1/64 (Table I I I ) . TABLE

III

A g g l u t i n a t i o n i n h i b i t i o n p a t t e r n s of a n t i - R h s e r u m w i t h a n t i - l d sera. ANTI-IDIOTYPIC SERA

Anti-Rh Serum LOR MOZ BRA BAU

CONTROL

LOR 4 sera

MOZ 2 sera

~G~ 2 sera

BRA 1 serum

BAU 2 sera

RNS

2.000

2.000

11ooo

1.000 1.0~ 1.0~ 1.0~ 1.0~

1.000 -1.000

2.000 1.000 1.000

6~T28

1.000 1.000

1.000

1.000

64-128 64-128 1.000

500 64 128

Partial inhibition was observed with heterologous anti-id sera. Sera w h i c h failed to detect cross-idiotypic specificities did not inhibit agglutination. To ensure that inhibition with anti-id sera was directed t o w a r d s the idiotypic d e t e r m i n a n t s of anti-Rh antibodies, we have tested these anti-id sera for their ability to inhibit a n o t h e r a n t i b o d y present in the serum. LOR s e r u m w h i c h contains anti-D and anti-C specificities was selected for this purpose. Anti-id LOR sera were f o u n d to agglutinate only coated R h + ( D + C--) RBC and not rh' RBC ( C + D--), although the latter were strongly agglutinated by antiglobulin sera. Anti-id LOR sera did not inhibit agglutination of rh' red cells by LOR serum. It could be suggested that a least p a r t of idiotopes are located in antigen-binding sites, i.e. the paratopes. F u r t h e r m o r e , failure of inhibition with anti-MOZ id sera suggested the existence of non antigen-binding idiotypic d e t e r m i n a n t s located on the f r a m e w o r k region of Rh antibodies. Similarly, it could be inferred that crossreactive idiotypes can be divided in antigen-binding and non-antigenbinding determinants. V. - - Follow-up of idiotypic specificities Agglutination titers and score of anti-id LOR sera for RBC coated by different samples of anti-Rh LOR sera, collected f r o m 1968 to 1977, were compared. Similar titers were obtained with the three anti-id sera against each of the different samples of coated RBC.

721

1DIOTYPY OF ANTI-Rh ANTIBODIES

The h i g h e s t t i t e r w a s o b s e r v e d a g a i n s t the RBC s e n s i t i z e d w i t h t h e 1973 a n t i b o d y . High t i t e r s w e r e also o b s e r v e d w i t h RBC s e n s i t i z e d w i t h 1968 a n d 1969 a n t i b o d i e s . The t i t e r s w e r e l o w e r a g a i n s t t h e 1970-1971 a n t i b o d i e s ( T a b l e I V ) . TABLE IV Agglutination titers of rabbit 603 serum against R h + RBC coated with samples of LOR Rh serum collected f r o m 1968 to 1977.

RBC coated with LOR serum

AGGLUTINATION

(1)

Titer (2)

Score (3)

1968 1969 1970 1971 1973

64 64 32 16 256

40 40 10 8

RBC coated with LOR serum

AGGLUTINATION

(1)

Titer (2)

Score (3)

1974 1975 1976 1977

128 64 64 16

51 24 24 17

60

Rabbit 603 has been injected with LOR antibodies purified from serum collected in 1973. The a g g l u t i n a t i o n - i n h i b i t i o n t e s t gave r e s u l t s w h i c h a r e in agreem e n t w i t h t h e a b o v e data. The h i g h e s t i n h i b i t i o n was given w i t h t h e s a m p l e s c o l l e c t e d in 1973 a n d 1974, b y c o n t r a s t low i n h i b i t i o n was given b y t h e 1970-1971 sera. VI.

--

EA-Rh

rosettes

They w e r e o b s e r v e d w i t h the l y m p h o c y t e s f r o m t h r e e donors, o b t a i n e d at d i f f e r e n t times, a n d a p p e a r e d 3 to 6 w e e k s a f t e r t h e b o o s t e r i n j e c t i o n of R h + RBC. They w e r e o b s e r v e d d u r i n g several m o n t h s a n d have d i s a p p e a r e d 7 m o n t h s a f t e r the b o o s t e r i n j e c t i o n in t h e case of a d o n o r i m m u n i z e d since 12 y e a r s . EA-Rh r o s e t t e s w e r e small, f o r m e d by 6 to 12 RBC s u r r o u n d i n g a l y m p h o c y t e . T h e i r a s p e c t w a s d i f f e r e n t f r o m t h a t of r o s e t t e s o b t a i n e d w i t h u n c o a t e d R h + r e d cells. The n u m b e r of EA-Rh r o s e t t e s w a s a l w a y s less t h a n 1% of t h e PBL. These r o s e t t e s c o u l d n o t be p e r f o r m e d w i t h l y m p h o c y t e s f r o m one D o n o r a n d R h + R B C s e n s i t i z e d w i t h Fab'2 f r a g m e n t s anti-Rh o b t a i n e d f r o m a n o t h e r Donor. The follow-up of the c o n c e n t r a t i o n of anti-Rh a n t i b o d y (t~g/ml) a n d of the n u m b e r of EA-Rh r o s e t t e s has s h o w n t h a t EA-Rh r o s e t t e s a r e a p p e a r i n g w h e n the c o n c e n t r a t i o n of the a n t i b o d y is d e c r e a s i n g

[28].

722

S A I N T M A R T I N (DE) et coll.

DISCUSSION

I t a p p e a r s f r o m o u r results t h a t the activity of the r a b b i t sera, o b t a i n e d b y i m m u n i z a t i o n w i t h purified anti-Rh antibodies, w a s - a f t e r suitable a b s o r p t i o n s - - s p e c i f i c for the Fab'2 or the anti-Rh antibody, p r o d u c e d b y a single d o n o r or a g r o u p of donors. This specific activity h a d anti-idiotypic characteristics, since it could not be detected w i t h a pool of h u m a n i m m u n o g l o b u l i n s or Fab'2 f r a g m e n t s f r o m n o r m a l individuals and was c o m p l e t e l y inhibited b y a b s o r p t i o n w i t h h o m o l o g o u s anti-Rh antibody. F u r t h e r m o r e , this activity was not d e p r e s s e d a f t e r contact w i t h anti-Rh s e r u m f r o m which the Rh antibodies h a d b e e n a b s o r b e d . These sera did not recognize allotopes or isotopes and they w e r e specific f o r idiotypic d e t e r m i n a n t s which are on or n e a r the p a r a t o p e s or on the framework. I t a p p e a r s f r o m o u r s t u d y t h a t specific idiotopes or idiotypes detected on the anti-Rh antibodies m a y be s h a r e d b y different antiRh donors. These results are in a g r e e m e n t w i t h those o b t a i n e d b y FORRE and al. [l 1], showing the existence of s h a r e d idiotypes on anti-Rh antibodies, a p p e a r i n g during i n c o m p a t i b l e pregnancies. These crossreacting idiotypes w e r e also dected in m o n o c l o n a l i m m u n o g l o b u l i n s , like cold agglutinins [10] a n d r h e u m a t o i d f a c t o r s [5]. By contrast, FEIz[ and al. h a v e s h o w n t h a t cross-idiotypic specificities are detected on h u m a n m o n o c l o n a l IgM w i t h anti-I specificity b u t are not s h a r e d b e t w e e n IgM anti-I and IgM anti-Pr [10]. Cross-reacting idiotopes w e r e not detected b y GEHA and WEINBERG [ 14] in the anti-tetanus antibodies p r o d u c e d b y t h r e e different Donors, t h o u g h the s a m e idiotopes w e r e s h a r e d b y the IgG, IgM and I g E of one i n d i v i d u a l . The c o m p l e x i t y of o u r results is p r o b a b l y related to the n u m b e r of individuals e x a m i n e d a n d to the length of the anti-Rh i m m u n i z a t i o n (3 to 10 years). No idiotypes or idiotopes w e r e s h a r e d b e t w e e n LOR anti-Rh antibodies a n d different anti-D antibodies p r o d u c e d b y 32 f r e n c h a n d 10 j a p a n e s e donors. Anti-D idiotypes or idiotopes are s h a r e d b y 13 of the 32 f r e n c h donors, using different s a m p l e s of 10 anti-idiotypic sera, o b t a i n e d b y i m m u n i z a t i o n of r a b b i t s w i t h antibodies f r o m 4 o t h e r donors. The idiotypic specificities detected on LOR antibodies are r e s t r i c t e d to the anti-D antibody. F u r t h e r m o r e , no cross-reacting idiotypes

IDIOTYPY OF ANTI-Rh ANTIBODIES

723

could be detected in antibodies specific for C, c, E, Kell and Duffy blood g r o u p antigens. FEIZI and a l [10] have s h o w n t h a t IgG anti-A belong mainly to the s u b g r o u p V H I, while anti-B belong to the sub-group VH I I I . Meanwhile, NATVIG and al have d e m o n s t r a t e d that anti-D belong to V H I I and anti-C to the V H I I I s u b g r o u p [21]. These results are indicating a restriction to the diversity of production of antibodies; the expression of the s a m e idiotopes by diff e r e n t individuals suggests that they are the p r o d u c t of the s a m e genes for the variable region. Cross-reacting idiotopes have b e e n o b s e r v e d in anti-DNA antibodies f r o m u n r e l a t e d patients [29, 31, 35]. The results o b t a i n e d by ZOUALI and one of us have s h o w n t h a t these idiotopes could be associated with antigen-binding site. By contrast, the results of SOLOMON and al. concerning u n r e l a t e d SLE patients are in favour of cross-reactive idiotopes non associated to the antigen-binding site. The data are suggesting that idiotope h u m a n r e p e r t o i r e of auto-antibodies is m o r e r e s t r i c t e d t h a n previously thought. The s a m e conclusions could be raised for Rh antibodies. Our d a t a showing t h a t different agglutination titers are obtained w i t h RBC sensitized w i t h different s a m p l e s of anti-Rh sera given b y a single donor, are in a g r e e m e n t w i t h the results o b t a i n e d b y OUDIN and MICHEL during the i m m u n i z a t i o n of r a b b i t s against S. typhi [24] a n d with the results o b t a i n e d b y BORDENAVE and OUDIN on the a p p e a r a n c e of n e w idiotypes in a follow-up of r a b b i t s i m m u n i z e d against S. abortus equi [3]. Similar o b s e r v a t i o n s w e r e r e p o r t e d for anti-p-amino-benzoate r a b b i t sera [20]. We suggest that in the case of the d o n o r LOR, the b o o s t e r injection of 1972 has s t i m u l a t e d the synthesis of idiotypes or idiotopes still detected five years later. These id could be p r o d u c e d de novo, r e p r e s e n t i n g thus an activation of new clones; however, they could be old-reappearing idiotypes which have b e e n extensively produced. The detection of small r o s e t t e s o b t a i n e d b y PBL s u r r o u n d e d b y R h + RBC sensitized with Fab'2 anti-Rh o b t a i n e d f r o m the d o n o r of lymphocytes, has s h o w n t h a t these r o s e t t e - f o r m i n g l y m p h o c y t e s are a p p e a r i n g at the t i m e of the decreasing of the anti-Rh antibodies. F o r instance, they w e r e detected 45 days a f t e r the b o o s t e r injection and w e r e still p r e s e n t 135 days after. The detection of cells able to stick to antibodies against virus, p r o t e i n s or haptens, is already known. F o r instance, TASlAUX a n d al. [3] have o b s e r v e d in r a b b i t s i m m u n i z e d against the virus of

724

SAINT MARTIN

(DE) et coll.

tobacco m o s a i c - - t h a t l y m p h o c y t e s of this type a p p e a r e d w h e n the antibodies' affinity was decreasing. In t h e case of o u r donors, the anti-idiotype activity of the lymp h o c y t e s is established as the r o s e t t e s w e r e n o t o b t a i n e d w i t h r e d cells coated w i t h the Fab'2 o b t a i n e d f r o m anti-Rh a n t i b o d y given by o t h e r donors. The increasing n u m b e r of the r o s e t t e - f o r m i n g l y m p h o c y t e s , able to recognize the idiotypes of the s a m e donor, is c o r r e l a t e d w i t h the decreasing a m o u n t of the antibodies. This suggests t h a t these lymp h o c y t e s could play a role in the regulation of the i m m u n e r e a c t i o n against the Rh antigen. These interactions of idiotype and anti-idiotype are playing a m a j o r role in the regulation of auto-immunity, a n d the defect is c o n s i d e r e d as an i m p o r t a n t p a r t of the pathogenesis of a u t o - i m m u n e diseases. A p p e a r a n c e of anti-idiotype against the anti-DNA a n t i b o d y has b e e n o b s e r v e d in the sera during inactive SLE a n d in n o r m a l individuals in contact w i t h these patients. These anti-idiotypes are not detected during the active p h a s e of SLE. The a p p e a r a n c e of auto-anti- idiotype seems to be linked to the decreasing activity of the disease [1]. The o c c u r r e n c e and evolution of auto-anti-idiotype antibodies to DNA h a s b e e n followed during 56 w e e k s b y ZOUALI a n d al. [34]. T h e r e w a s good c o r r e l a t i o n b e t w e e n the levels of a n t i d d i o t y p e antibodies a n d the periods of the disease. The variations of the p r e s e n c e of the idiotopes o b s e r v e d on the LOR Rh antibodies are fitting well w i t h this picture. The b o o s t e r injection of R h + red cells is giving a s t i m u l a t i o n of n e w clones p r o d u c i n g Rh antibodies w i t h new idiotopes, which are stimulating the p r o d u c t i o n of new anti-id b y s o m e o t h e r clones. However, the s u p p r e s s i o n could be m o r e or less effective on certain clones and could allow s o m e of t h e m to be p r e p o n d e r a n t . This type of a u t o - s u p p r e s s i o n is p r o b a b l y defective during autoi m m u n e diseases k n o w n to be associated with high titers autoantibodies. RESUME Des sdrums anti-idiotypiques ont 6t6 o b t e n u s p a r i m m u n i s a t i o n de lapins avec des a n t i c o r p s anti-Rh purifi6s p a r dlution. Les a n t i c o r p s de 5 d o n n e u r s de sang ont 6t6 s61ectionn6s p o u r cette dtude. Apr6s les a b s o r p t i o n s appropri6es, ces sdrums anti-idiotypiques agglutinent, h des titres 61ev6s, des globules rouges Rh positifs, sensibilis6s p a r les a n t i c o r p s du s 6 r u m a y a n t servi ~ l ' i m m u n i s a t i o n .

1 D I O T Y P Y OF ANT1-Rh A N T I B O D I E S

725

Certains d'entre eux agglutinent 6galement, mais avec des titres moins 61ev6s, les globules rouges Rh positifs, sensibilis6s par des anticorps de quelques-uns d ' u n groupe de 32 donneurs. L'existence d'idiotypes ou de d6terminants idiotypiques c o m m u n s aux anticorps de plusieurs sujets n o n apparent6s, a 6galement 6t6 d6montr6e p a r inhibition de l'h6magglutination. Une partie des idiotypes mis en 6vidence sont localis6s dans le paratope, ou h son voisinage imm6diat, alors que d'autres sont situ6s dans le " f r a m e w o r k " . De plus, nous avons pu suivre, sur une p6riode de 9 arts, la persistance et l'6volution des sp6cificit6s idiotypiques des anticorps du d o n n e u r LOR. Les rdsultats m o n t r e n t que des sp6cificit6s idiotypiques peuvent disparaitre ou apparaitre, ou encore devenir prdponddrantes aux d6pens de certaines autres. Certains lymphocytes du sang p6riph6rique p o r t e n t des r6cepteurs p o u r les Fab'2 d'anticorps anti-Rh a p p a r t e n a n t au m~me donneur. Ces cellules peuvent 6tre raises en 6vidence, d~s que le taux d'anti. corps anti-Rh c o m m e n c e h diminuer, dans les semaines suivant l'injection de rappel. SUMMARY Anti-id sera to Rh antibodies were p r o d u c e d by injecting rabbits with purified Rh antibodies. These sera were s h o w n to agglutinate O R h + RBC coated by the immunizing antibody a n d - - i n some c a s e s - - b y other anti-D antibodies. I d and cross-reactive id were shown to be located in the antigen-binding and in the non-antigen binding regions of Rh antibodies. An unique example of evolution of idiotypic specificities on h u m a n antibodies has been reported. Lastly, we have d e m o n s t r a t e d by rosette assay, presence on some PBL of receptors for Fab'2 anti-Rh coating O R h + red cells. Rosettes could not be obtained with l y m p h o c y t e s of a d o n o r and Fab'2 antiRh of a n o t h e r individual. Rosettes appeared at a period of time in which the a m o u n t of antibody was decreasing.

A b b r e v i a t i o n s u s e d in this paper:

Anti-id = Anti-idiotypic -- Ig = Immunoglobulins -- PBL -"Peripheral blood lymphocytes R = Rabbit -- RBC = Red blood cell RNS = Rabbit normal serum -- SLE = Systemic lupus erythematosus -

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Request reprints from: J. DE SAINTMARTIN, Institut Pasteur, Rue du Dr-Roux, 75015 PARIS.

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REFERENCES [1] ABDOU N.I., WALL H., LINDSLEY H.B., HALSEY J.F. and SUZUKI T. - Network theory in a u t o i m m u n i t y . In vitro suppression of s e r u m antiDNA antibody b i n d i n g to DNA by anti-idiotypic a n t i b o d y in systemic lupus erythematosus. J. Clin. Invest., 67, 1297, 1981. [2] BOCCADORO M., VAN ACKER A., PILORI A. a n d URBAIN J. - - Idiotypic lymphocytes ill h u m a n monoclonal gammapathie~. Ann. ImmunoI. (Inst. Pasteur), 132 C, 9, 1981. [3] BORDENAVE G. et OUOIN J. - - L'idiotypie des anticorps de lapin antiSalmonella abortus equi. I. Les r~actions h~t6rologues. Ann. Inst. Pasteur, 120, 265, 1971. [4] BOYUMA. - - Isolation of lymphocytes, granulocytes and macrophages. Scand. J. ImmunoI., 5 (suppl. 5), 9, 1976. [5] CARSOND.A. and FONG S. - - A c o m m o n idiotope on h u m a n r h e u m a t o i d factors identified by a h y b r i d o m a antibody. Molec. Immunol., 20, 1081, 1983. [6] CUNNINGHAM-RUNDLES C. - - Naturally occurring autologous anti-idiotypic antibodies. Participation in i m m u n e complex f o r m a t i o n in selective IgA deficiency. ]. exp. Med., 155, 711, 1982. [7] DELLAGI K., BROUET J.C. a n d DANON F. - - Cross-idiotypic antigens a m o n g monoclonal i m m u n o g l o b u l i n M from patients with WaldenstrSmJs m a c r o g l o b u l i n e m i a a n d polyneuropathy. J. Clin. Invest., 64, 1530, 1979. [8] DOBLOUG J.H., FORRE O., NATVIG J.B. a n d MICHAELSEN T.E. - - Dem o n s t r a t i o n of r h e u m a t o i d factor idiotypic antigens on peripheral blood B a n d T lymphocytes from patients with r h e u m a t o i d arthritis. Scand. J. Immunol., 9, 273, 1979.

[9] EBERS G.C., ZABRISKIE J.B. and KUNKEL H.G. - - Oligoclonal i m m u n o globulins in subacute sclerosing panellcephalitis a n d multiple sclerosis: a study of idiotypic d e t e r m i n a n t s . Clin. Exp. ImmunoI., 35, 67, 1979. [10] FEIZI T., KUNKEL H.G. and ROELCKE D. - - Cross idiotypic specificity a m o n g cold agglutillins in relation to c o m b i n i n g activity for blood group-related antigens. Clin. Exp. Immunol., 18, 283, 1974. [11] FORRE O., GAARDER P.I. a n d NATVIG J.B. - - Va subgroup restriction in h u m a n erythrocyte antibodies: Studies of anti-A, anti-B a n d antiDuffy antibodies. Scand. ]. Immunol., 6, 149, 1977. [12] FRANKLINE.C. a n d FRANGIONEB. - - C o m m o n s t r u c t u r a l a n d antigenic properties of h u m a n , ~M anti-y-globulins. J. Immunot., 107, 1527, 1971. [13] GEHAR.S. - - Presence of auto-anti-idiotypic a n t i b o d y during the n o r m a l h u m a n i m m u n e response to t e t a n u s toxoid antigen. J. Immunol., 129, 139, 1982. [14] GEHA R.S. and WEINBERG R.P. - - Anti-idiotypic a n t i s e r a in man. I. Production a n d i m m u n o c h e m i c a l characterization of allti-idiotypic anti-sera to h u m a n a n t i t e t a n u s antibodies. J. Immunol., 121, 1518, 1978.

IDIOTYPY OF ANT1-Rh ANTIBODIES

727

[15] GELTNER D., FRANKLIN E.C. a n d FRANGIONEB. - - Anti-idiotypic activity in the IgM fractions of mixed cryoglobulins. J. ImmunoI., 125, 1530, 1980. [16] KENNEDY R.C. and DREESMAN G.R. - - C o m m o n idiotypic d e t e r m i n a n t associated with h u m a n antibodies to hepatitis B surface antigen. J. Immunol., 130, 385, 1983. [17] KUNKEL H.G., MANNIK M. a n d WILLIAMS R.C. - - Individual antigenic specificity of isolated antibodies. Science, 140, 1218, 1963. [18] LEFVERT A.K. - - Anti-idiotypic antibodies against the receptor antibodies in m y a s t h e n i a gravis. Scand. J. ImmunoI., 13, 493, 1981. [19] MATSUYAMA T., FUKUMORI J. and TANAKA H. Evidence of unique idiotypic d e t e r m i n a n t s a n d similar idiotypie d e t e r m i n a n t s on h u m a n antithyroglobulin antibodies. Clin. Exp. Immunol., 51, 381, 1983. [20] McDONALD A.B. a n d NISONOEF A. - - Quantitative investigations of idiotypic antibodies. III. Persistence and variations of idiotypic speeificities during the course of immunizations. J. Exp. Med., 131, 583, 1970. [21] NATVIG J.]3., FORRE O. and MICHAELSEN T.E. - - Restriction of h u m a n i m m u n e antibodies to heavy-chain variable subgroups. Scan& J. ImmunoL, 5, 667~ 1976. [22] NATVIGJ.B., KUNKEL H.G., ROSENFIELDR.E., DALTONJ.F. a n d KOCHWA S. - - Idiotypic specificities of anti-Rh antibodies. J. ImmunoL, 116, 1536, 1976. [23a] OUDINJ. et MICHEL M. - - Une nouvelle forme d'allotypie des globulines du s6rum de lapin, a p p a r e m m e n t lide h la fonction et g la sp6cificit6 anticorps. C.R. Acad. Sci. (Paris), 257, 805, 1963. [23b) OUDIN J. - - Genetic regulation of i m m u n o g l o b u l i n synthesis. J. cell. Physiol., 67 (suppl. 1), 77, 1966. [24] OUDIN J. and MICHEL M. - - Idiotypy of r a b b i t antibodies. II. Comparison of idiotypy of various kinds of antibodies formed in the same rabbits against Salmonella typhi. J. Exp. Med., 130, 619, 1969. -

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[25] PREUD'HOMMEJ.L., KLEIN M., LABAUMES., a n d SELIGMANNM. - - Idiotypebearing a n d antigen-binding receptors produced by blood T lymphocytes in a case of h u m a n myeloma. Eur. J. Immunol., 7, 840, 1977. [26] SAINT MARTIN ]'. DE, MUKOYAMAH. et EYQUEM A. - - Evolution p e n d a n t 9 ads de l'idiotypie des anticorps anti-Rh chez u n D o n n e u r de sang. C.R. Acad. Sci. (Paris), 286, 1183, 1978. [27] SAINT MARTIN J. DE, MUKOYAMA H. et EYQUEM A. - -

R6actions h6t6rologues observ6es h l'6tude de l'idiotypie des anticorps anti-Rh. Ann. Immunol. (Inst. Pasteur), 129 C, 731, 1978. [28] SAINT MARTIN J. DE, SCHWARTZ J., MANNESSIER L. and EYQUEM A. - Idiotypic and anti-idiotypie d e t e r m i n a n t s on lymphocytes during antiRh i m m u n i z a t i o n . Blood Transl. and Immunohaematol., 26, 573, 1983. [29] SHOENFELD Y., ISENBERG D.A., RAUCH J., MADAIO M.P., STOLLAR B.D. a n d SCHWARTZ R.S. Idiotypic cross-reactions of monoclonal h u m a n lupus autoantibodies. J. Exp. Med., 158, 718, 1983. [30] SLATER R.J., WARD S.M. a n d KUNKEL H.G. - - I m m u n o l o g i c a l relationships among the m y e l o m a proteins. J. Exp. Med., 101, 85, 1955. -

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728

SAINT MARTIN (DE) et coll.

J., KEISER H . D . and DIAMOND B. - - Use of m o n o c l o n a l a n t i b o d i e s to identify s h a r e d idiotypes on h u m a n antibodies to native DNA f r o m p a t i e n t s w i t h systemic lupus e r y t h e m a t o s u s . Proc. Nat. Acad. Sci (Wash.), 80, 850, 1983. TASIAUX N . , LEUWENKROON R., BRUYNS C. a n d URBAIN ft. - - Possible o c c u r r e n c e a n d m e a n i n g of l y m p h o c y t e s b e a r i n g autoanti-idiotypic r e c e p t o r s d u r i n g the i m m u n e response. Eur. J. IrnrnunoL, 8, 464, 1978. WILLIAMS R.C., KUNKEL H.G. a n d CAPRA J.I). - - Antigenic Specificities r e l a t e d to the cold agglutinin activity of g a m m a M globulins. Science, 161, 379, 1968. ZOUALI M. a n d EYQUEM A. - - I d i o t y p i c / a n t i - i d i o t y p i c interactions in systemic lupus e r y t h e m a t o s u s : d e m o n s t r a t i o n of oscillary levels of anti-DNA a u t o a n t i b o d i e s a n d r e c i p r o c a l anti-idiotypic activity in a single patient. Ann. I m m u n o l . (Inst. Pasteur), 134 C,, 377, 1983. ZOUALI M. a n d EYQUEM A. J I d i o t y p i c / a n t i - i d i o t y p i c i n t e r a c t i o n s in h u m a n a n t o a n t i b o d i e s to DNA in systemic lupus e r y t h e m a t o s u s . I m m u n o l o g y Lelters, 7, 187, 1984.

[31] SOLOMON G., SCHIFFENBAUER

[32]

[33]

[34]

[34]