Perinatally Derived Antiidiotypic Antibodies from the Antipolyfructosan Immune Response Molecular Analysis of Idiotype Regulation" JOACHIM H. BARTELS, MEENAL ELLIOTT, AND JOHN F. KEARNEY Division of Developmental and Clinical Immunology Department of Microbiology University of Alabama at Birmingham Birmingham, Alabama 35294

Newborn mice and humans are unable to mount an immune response against bacterial surface antigens like phosphorylcholine, a 1-3 dextran, and the polyfructosans inulin and levan. Naturally occurring antibodies derived from perinatal BALB/c mice are multireactive and display a high frequency of antiidiotypic (anti-id) antibodies specific for the major antipolyfructosan idiotype J606. The hypothesis that the J606 idiotype is downregulated by anti-id antibodies early in development is supported by the observation that injection of anti-id antibodies during certain times early in development results in a further delay of the ability to mount an antipolyfructosan immune response, which normally occurs 3-4 weeks after birth in BALB/c mice. More generally these and other observations support the hypothesis that in the early neonatal period complex interactions between germ-line encoded complementary sets of IgM B-cell V-region determinants constitute a driving force in the establishment of the B-cell repertoire. To study the molecular nature of idiotype/antiidiotype interactions proposed to shape the development of antipolysaccharide immune responsiveness during ontogeny, variable regions of heavy (V,) and light (V,) chains of natural IgM anti-id antibodies specific for antipolyfructosan antibodies (J606 idiotype) were sequenced from PCRamplified cDNA derived from BALB/c fetal and neonatal hybridoma RNA. The V gene segments from seven hybridomas sequenced (TABLE1) were found to be of germ-lineor presumed germ-line origin and represented five V, gene families (VH36-60, VH9, VHIO, V,Q52, and 3 * VH7183), thus showing a bias towards D proximal gene families. Two of the V,7183 monoclonal antibodies (mAb) used the 81X gene, a V,-gene functionally expressed only early in development, not in the adult. D-segments DFL16-1 (2/7) rearranged to J,1 and DQ52 (3/7) rearranged to JH2were used preferentially. The third heavy chain complementarity-determining region (VH-CDR3) lacked N-segment insertion, and reading frame utilization of the D segment was limited. Preferential usage of certain points of recombination at the D-J junction was apparently predetermined by sequence homology between D and J germ-line genes. Because the lack of N regions in perinatal B cells is not compensated for by deletion of fewer regions nucleotides from coding regions of D and J segments, the perinatal VH-CDR~ 'This work was supported by NIH Grants A114782 and AI30879. 316

fetal liver (16-day embryo) fetal liver (2 days before birth) fetal liver (2 days before birth) liver (2 days after birth) liver (2 days after birth) liver (2 days after birth) liver (2 days after birth) spleen (adult) vH

v,

P3K , A p, K, A

f4

P 9

K

K

P,K

V, 7183

P,K 1

JH2 JH2 JH3

DQ52 DQ52 & DSP2.8

452 J558

VH 36-60

DQ52

JH3

JH

JH1

Jd

JH

J H ~

b

DFL 16.1

DFL 16.1

DSP 2.10b

D

DQ52

10

VH 7183

V, 7183

VH

P*K

P. K

Isotype

Heavy chain

1

ND

JK4

VK4 ND

J2 v,9

ND

J,5/Jk1

N D f l kI N D

J,

1

J,5

JK

J,/J,

V,12-13

V,12-I 3

K '9

v,fl,

Light chain

a V-DJ usage of heavy and V-J usage of light chains. V regions from heavy and light chains of seven anti-id antibodies (7 perinatally derived, 1 from an adult mouse) were sequenced. Perinatally derived antibodies sequenced here seem to preferentially use genes from DHproximal gene families (VH7183 or VHQ52). New D segments, only recently described. ND, not determined.

E F 1-33

GA6-4

AB 5

LA1-9

34- 1

ED6

BC2-5

16-165

Source

Summary of Sequencing Results"

Hybridoma

TABLE 1.

w

5

%

E2

8

B

3

3

Ei

n P

318

ANNALS NEW YORK ACADEMY OF SCIENCES

are often shorter than those of the adult. This reduction in length, the observed reduced V,-CDR3 variability, and the use of germ-linegenes indicate that the perinatal antibody population differs from the adult antibody repertoire structurally and functionally with respect to range of idiotopes and specificities. Gene-bank search revealed other antibodies that share V,-CDR3 regions similar or identical to those found here emphasizing the general nature of the mechanisms that limit V,-CDR3 variability and that have the potential to create idiotopes common to otherwise different antibodies: mAb ED6 recognizes mAbs GA6 and LA1-9, which share an almost identical V,-CDR3 region; LA1-9 inhibits ED6 binding to GA6, and GA6 inhibits ED6 binding to LA1-9, indicating that ED6 may recognize the VH-CDR3, a structure common to both LA1-9 and GA6. We propose that localized sequence similarities between V,-CDR3 regions, as well as between other germ-line encoded V-region segments, may be responsible for idiotopes shared between these molecules and be associated with the multireactivity typical of perinatal antibodies. A peptide based on V,-CDR2 sequence similarity between J606 and an antiJ606 antibody was found to interact with the majority of antiJ606 antibodies and was able to inhibit self-binding of the anti-id antibody containing this V,-CDRZ sequence as well as other idiotypic interactions involving J606 or LA1-9. The VH-CDR2-derived peptide and other peptides based on shared idiotopes may be used as vaccines in providing a tool to manipulate the immune response of neonates to polysaccharide antigens.