Predominance of VH-D-JH junctions occurring at sites of short sequence homology results in limited junctional diversity in neonatal antibodies.

Sequence diversity at the junctions of Ig genes differs between newborn and adult mice in two respects: 1) fetal/newborn Ig lack N regions; and 2) these N- junctional sequences very often contain 1 to 6 nucleotides that could have been encoded by either of the two joined gene segments. We address the hypothesis that such short homologies preferentially direct recombination to that site, and we analyze the effect of such homology-directed recombination upon the neonatal Ig repertoire. We examined 546 CDR3 sequences that were generated from polymerase chain reaction-amplified DNA from fetal and newborn liver using primers from three different VH families: S107, 7183, and J558. All junctional sequences using 14 frequently occurring IgH V-D and D-J gene combinations were analyzed. In 12 of the 14 combinations analyzed, there were 1 to 3 short sequence homologies, and the junctional sequences that would be created by those homologies were observed with high frequency. The D-J junctions often had two to three predominant junctional sequences, whereas the V-D junctions had one dominant junctional sequence. The only exceptions were the VHJ558-D junctions, where homology-directed recombination using the sequence homology between VHJ558 genes and most D genes would result in an out-of-frame join, and most of our sequences were productive. This latter result further suggests that homology-directed recombination may play a role in the nonrandom VH gene usage observed in fetal and newborn mice. Thus, most neonatal IgH junctions show limited diversity, not only due to the lack of N regions, but also because of nonrandom junctional sequences. Inasmuch as the few adult N- junctions also show a high frequency of homology-directed junctional sequences, V-D-J recombination throughout life may involve pairing via short homologies, with addition of N regions obscuring its role in the formation of adult IgH junctions.     

Germ-line sequence of the DH segment employed in Ars-A antibodies: implications for the generation of junctional diversity

The majority of antibodies produced by A/J mice in response to p-azophenylarsonate belong to the Ars-A family. These antibodies have the conserved sequence cys-ala-arg-ser-x-tyr-tyr (in which x is variable) spanning the V-D junction of the heavy chain. The cys-ala-arg residues are accounted for in the sequence of the A/J VH gene; the tyr-tyr are believed to be specified by the A/J DH segment, although this assumption is based on the DFL16.1 sequence derived from BALB/c mice. This implies that the ser-x is generated by joining imprecision and/or N segment addition. More recent data have revealed that the codon specifying the junctional serine residue is highly conserved (TCN, where N is usually G), suggesting a germline origin. Because there is no obvious way to generate this codon from the A/J Ars-A VH gene, we examined the involvement of the A/J DH segment in the generation of this junctional residue by cloning and sequencing the A/J equivalent to DFL16.1. We have established that this DH segment is polymorphic among BALB/c and A/J at the nucleic acid sequence level, and that it does not encode the junctional serine. This implies that a mechanism other than joining imprecision or random N segment addition operates at V-D junctions of Ars-A heavy chains.     

Unusual germline DSP2 gene accounts for all apparent V-D-D-J rearrangements in newborn, but not adult, MRL mice.

Anti-dsDNA autoantibodies in MRL mice contain a higher than average frequency of atypical complementarity-determining regions 3, including those made with D-D rearrangements. It has been reported that MRL mice have an intrinsically high frequency of creating VDDJ rearrangements; however, we show in this study that the majority of these apparent D-D rearrangements in B cell progenitors can be accounted for by a very novel germline D(H) gene in mice of the Igh(j) haplotype. This gene has the appearance of a D to D rearrangement due to the duplication of 9 bp common to most DSP2 genes. Germline DSP2 genes from Igh(j) mice were amplified, cloned, and sequenced, showing the presence of this novel gene as well as a new allele of a conventional DSP2 gene. Sequencing of D-J rearrangements revealed that Igh(j) mice also have a different allele of DFL16.1 and apparently lack DFL16.2. Despite the existence of this new DSP gene, analysis of VDJ rearrangements from adult bone marrow pre-B cells of MRL/lpr mice still revealed the presence of complementarity-determining region 3 containing apparent D-D joinings in 4.6% of the sequences. C3H pre-B cells had 4.2% of sequences with apparent VDDJ rearrangements, and BALB/c pre-B cells had approximately 2%. DDJ intermediates were also observed, but at a lower frequency. However, strikingly, no VDDJ rearrangements were observed in newborn sequences, suggesting the process of assembly of VDJ rearrangements is fundamentally different in newborn mice vs adult mice.     

Perturbation of the idiotypic network. II. Transfer of suppression to neonatal mice

The cellular mechanisms which are responsible for idiotype perturbation induced by repeated stimulation with either allogeneic mixed lymphocyte culture-generated syngeneic blasts or allogeneically stimulated syngeneic spleen cells were investigated as described in the preceding article. Using the splenic fragment culture system, the precursor frequencies of T and B cells for anti-phosphorylcholine (PC) antibodies and T15 idiotypic antibodies were determined in allogeneically challenged mice. Adoptive transfers of T cells to neonatal BALB/c mice induced suppression of the T15+ anti-PC responses. In addition, the effect of immunization with internal image-bearing anti-idiotopes on the level of anti-PC antibodies and T15 idiotype was determined. Results from this study demonstrated (i) a decrease in the precursor frequency in the PC-specific and idiotype-specific B cell repertoire; (ii) a decrease in the precursor frequency of T helper cells, which recognize idiotypes and anti-idiotypes; (iii) the possibility to transfer T15 suppression to neonatal mice; and (iv) the possibility to restore T15 dominance by anti-idiotypic antibody immunization. These data indicate that both the T and B compartments are involved in the maintenance of suppression induced by repeated exposure to alloantigen-sensitized syngeneic cells. Collectively these findings show that a nonspecific general suppression induced by allohyperimmunization can perturb the T15+ anti-PC response.     

Sequence homologies, N sequence insertion and JH gene utilization in VHDJH joining: implications for the joining mechanism and the ontogenetic timing of Ly1 B cell and B-CLL progenitor generation.

Sequence analysis of rearranged VHDJH genes of B lineage cells from various stages of ontogeny indicates that short sequence homologies at the breakpoints of recombination contribute to V region gene assembly. Such homologies are regularly seen at DJH junctions of neonatal pre-B cells, most of which do not contain N sequences. In the same cells, but not at later developmental stages, preferential usage of the JH1 element is observed. After birth, N sequence insertion increases with time and is always more prominent at the VHD border than the DJH border. In pre-B cells from adult animals and in mature B cells, in cases where N sequences were not detectable, sequence homologies at the DJH border were found in only half of the instances. This lower incidence could be due to N sequence addition to one of the recombining DNA ends and/or cellular selection. Inspection of VHDJH junctions for N sequence insertion, sequence homologies at the DJH border and JH1 usage allows the estimation of the timepoint in ontogeny at which particular B cell subsets are seeded into the immune system. Specifically, the present data show that the cells of the Ly1 B cell subset are generated not only neonatally but also beyond the first weeks of life. However, the DJH junctions of the progenitors of chronic B cell leukemias which originate from the same B cell subset resemble those of neonatal pre-B cells, suggesting that these cells have already undergone a transforming event at this early developmental stage.     

Specific VDJ gene combinations contribute to the specificity of memory antibodies to the phosphorylcholine hapten.

Based on their fine specificity, two groups of antibodies against the phosphorylcholine (PC) hapten have been described. Group I antibodies react predominantly with the PC moiety of the hapten and group II are directed against the entire hapten including the azophenyl spacer to the protein carrier. We have analyzed the VH gene segment utilization of hybridomas from the memory response to PC by Southern blot analysis and nucleotide sequencing of the functional VDJ rearrangements. Three main specificities of anti-PC antibodies could be distinguished. Anti-PC hybridomas with group I fine specificity utilize the VH1-DFL 16.1-JH1 rearrangement. A major portion of group II antibodies recognized the phenyl-PC part and expressed the same VH1 gene in combination with a member of the SP2 family and JH1 or JH2. The other anti-PC antibodies either used the PJ14-DFL16-JH3 rearrangement in combination with a lambda 1 L chain or a member of the VGam3.8 VH family rearranged to the DFL16.1 and the JH3 gene segments. The PJ14 and VGam3.8 V gene expressing antibodies were directed to the phenyl group and were either not or barely inhibitable by PC chloride. Thus, specific VDJ gene combinations contribute to the fine specificity of antibodies in the memory response to the PC hapten. The use of the S107, Q52, and VGam3.8. VH gene families, together with FL16.1 or SP2 D segments and JH1, JH2, or JH3 results in different fine specificities to the PC, phenyl-PC, or the azophenyl moiety of the PC hapten. These fine specificities of the memory response use V, D, and J segments of the initial T15Id+ response in combination with gene segments usually related to phenyl specificity.     

P nucleotides in V(D)J recombination: a fine-structure analysis.

Antigen receptor genes acquire junctional inserts upon assembly from their component, germ line-encoded V, D, and J segments. Inserts are generally of random sequence, but a small number of V-D, D-J, or V-J junctions are exceptional. In such junctions, one or two added base pairs inversely repeat the sequence of the abutting germ line DNA. (For example, a gene segment ending AG might acquire an insert beginning with the residues CT upon joining). It has been proposed that the nonrandom residues, termed "P nucleotides," are a consequence of an obligatory end-modification step in V(D)J recombination. P insertion in normal, unselected V(D)J joining products, however, has not been rigorously established. Here, we use an experimentally manipulable system, isolated from immune selection of any kind, to examine the fine structure of V(D)J junctions formed in wild-type lymphoid cells. Our results, according to statistical tests, show the following, (i) The frequency of P insertion is influenced by the DNA sequence of the joined ends. (ii) P inserts may be longer than two residues in length. (iii) P inserts are associated with coding ends only. Additionally, a systematic survey of published P nucleotide data shows no evidence for variation in P insertion as a function of genetic locus and ontogeny. Together, these analyses establish the generality of the P nucleotide pattern within inserts but do not fully support previous conjectures as to their origin and centrality in the joining reaction.     

Partial versus productive immunoglobulin heavy locus rearrangements in chronic lymphocytic leukemia: implications for B-cell receptor stereotypy

The frequent occurrence of stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences among unrelated cases with chronic lymphocytic leukemia (CLL) is widely taken as evidence for antigen selection. Stereotyped VH CDR3 sequences are often defined by the selective association of certain immunoglobulin heavy diversity (IGHD) genes in specific reading frames with certain immunoglobulin heavy joining (IGHJ ) genes. To gain insight into the mechanisms underlying VH CDR3 restrictions and also determine the developmental stage when restrictions in VH CDR3 are imposed, we analyzed partial IGHD-IGHJ rearrangements (D-J) in 829 CLL cases and compared the productively rearranged D-J joints (that is, in-frame junctions without junctional stop codons) to (a) the productive immunoglobulin heavy variable (IGHV )-IGHD-IGHJ rearrangements (V-D-J) from the same cases and (b) 174 D-J rearrangements from 160 precursor B-cell acute lymphoblastic leukemia cases (pre-B acute lymphoblastic leukemia [ALL]). Partial D-J rearrangements were detected in 272/829 CLL cases (32.8%). Sequence analysis was feasible in 238 of 272 D-J rearrangements; 198 of 238 (83.2%) were productively rearranged. The D-J joints in CLL did not differ significantly from those in pre-B ALL, except for higher frequency of the IGHD7-27 and IGHJ6 genes in the latter. Among CLL carrying productively rearranged D-J, comparison of the IGHD gene repertoire in productive V-D-J versus D-J revealed the following: (a) overuse of IGHD reading frames encoding hydrophilic peptides among V-D-J and (b) selection of the IGHD3-3 and IGHD6-19 genes in V-D-J junctions. These results document that the IGHD and IGHJ gene biases in the CLL expressed VH CDR3 repertoire are not stochastic but are directed by selection operating at the immunoglobulin protein level.     

V gamma 3 T cell receptor rearrangement and expression in the adult thymus.

Rearrangement and expression of the V gamma 3-J gamma 1 TCR has been found in murine dendritic epidermal cells (DEC) and fetal thymus. By using the polymerase chain reaction technique, V gamma 3-J gamma 1 rearrangements and RNA expression were detected in the murine adult thymus. Individual genomic and cDNA junctions were cloned and sequenced. In genomic DNA, 55% (16/29) of V gamma 3-J gamma 1 junctional sequences had N regions ranging in length from 1 to 12 nucleotides resulting in considerable junctional diversity. Only 5% (2/42) of cDNA sequences had N regions. The canonical DEC sequence represented 36% (15/42) of the cDNA sequences. Thus, fetal-type V gamma 3-J gamma 1 rearrangements lacking N regions were preferentially expressed in adult thymocytes, some of which may be DEC precursors. The developmental stages in which V gamma 3-J gamma 1 rearrangements are generated were studied by using polymerase chain reaction to detect circular rearrangement products. Active V gamma 3-J gamma 1 rearrangement was detected in thymuses from fetal, newborn, and 2-wk-old mice but not in 5-wk or 8-wk-old (adult) mice. V gamma 2, one of the most common V gamma rearrangements in the adult, was found to be actively rearranging to J gamma 1 in the adult thymus. However, V gamma 2-V gamma 3 replacement rearrangement was not found. These results support the hypotheses that adult thymocytes with rearranged V gamma 3-J gamma 1 are persistent from earlier developmental stages and represent a separate lineage from those with V gamma 2-J gamma 1 rearrangements.     

Sequence and fine specificity analysis of primary 511 anti-phosphorylcholine antibodies

The primary antibody response of mice to phosphorylcholine (PC) is dominated by antibodies using the T15 L chain. Anti-PC antibodies using the 511 L chain are prominent only in secondary responses to PC coupled to proteins, are somatically mutated, and all have an extra amino acid at the Vh-D junction, compared with T15 antibodies. The aim of the experiments reported here was to determine if the extra junctional amino acid alone was sufficient to generate a 511 PC-binding antibody, or if somatic mutation or other junctional changes were also necessary. We also wished to determine if unmutated 511 antibodies had sufficient affinity for PC to appear in the primary response. To increase the frequency of primary 511 antibodies, we generated a series of hybridomas from M167 L chain transgenic mice immunized 4 days earlier with either Streptococcus pneumonia R36a or PC-keyhole limpet hemocyanin (KLH). We determined the relative affinity of the antibodies, and sequenced their H chain V regions. The results showed that: 1) somatic mutations are not required for 511 antibodies to bind PC; 2) primary 511 antibodies all had lower relative affinities for PC than T15 while having similar affinities to T15 for TNP-aminophenyl PC, and higher affinities for the PC analogs nitrophenyl PC and choline; 3) all antibodies had the 511-specific insertion of an extra amino acid, usually Ala, at the VhD junction, compared with T15; 4) immunization with R36a, but not PC-hemocyanin, elicited antibodies with a specific Tyr----Asp substitution in the D region, indicating Ag selection based on fine specificity differences; 5) the total length of CDR3 was conserved in most anti-PC-hemocyanin antibodies, whereas the anti-R36a antibodies predominantly had longer CDR3 sequences; and 6) there were unique substitutions in most antibodies, including significant sequence heterogeneity in the D-Jh junction. We conclude that Ag selection on the basis of affinity for PC biases the primary anti-PC response in favor of T15, and that 511 precursors with their alternative fine specificities contribute the precursors that are expanded in the secondary anti-PC-KLH responses.     

A joining-diversity-joining complex generated by inversion mechanism and a variable-diversity complex in the beta-chain gene of the human T-cell receptor.

We have analysed an inactive allele of the beta-chain gene of the T-cell receptor in a human T-cell line HPB-ALL. Comparison with germline sequences showed that HPB-ALL has a joining (J)-diversity (D)-J complex recombined in head-to-head configuration and a variable (V)-D complex in tail-to-tail configuration. These results demonstrate that the inversion mechanism functions in the beta-chain gene of the T-cell receptor. The presence of the V-D complex suggests that V-D recombination could occur prior to D-J recombination although there is no definite proof that the V-D complex is an intermediate to form the V-D-J complex.     

Palindromic Nucleotide Analysis in Human T Cell Receptor Rearrangements

Diversity of T cell receptor (TCR) genes is primarily generated by nucleotide insertions upon rearrangement from their germ line-encoded V, D and J segments. Nucleotide insertions at V-D and D-J junctions are random, but some small subsets of these insertions are exceptional, in that one to three base pairs inversely repeat the sequence of the germline DNA. These short complementary palindromic sequences are called P nucleotides. We apply the ImmunoSeq deep-sequencing assay to the third complementarity determining region (CDR3) of the β chain of T cell receptors, and use the resulting data to study P nucleotides in the repertoire of naïve and memory CD8⁺ and CD4⁺ T cells. We estimate P nucleotide distributions in a cross section of healthy adults and different T cell subtypes. We show that P nucleotide frequency in all T cell subtypes ranges from 1% to 2%, and that the distribution is highly biased with respect to the coding end of the gene segment. Classification of observed palindromic sequences into P nucleotides using a maximum conditional probability model shows that single base P nucleotides are very rare in VDJ recombination; P nucleotides are primarily two bases long. To explore the role of P nucleotides in thymic selection, we compare P nucleotides in productive and non-productive sequences of CD8⁺ naïve T cells. The naïve CD8⁺ T cell clones with P nucleotides are more highly expanded.     

Differential L chain expression in the antibody responses to phosphorylcholine of adult bone marrow or peritoneum-derived B lymphocytes

Lethal irradiation of adult BALB/c mice followed by reconstitution with autologous bone marrow results in loss of T15 Id and IdX expression in the responses to phosphorylcholine (PC) and alpha(1-3)-dextran, respectively. T15 Id, but not IdX expression can be reconstituted with low numbers of syngeneic, T cell-depleted peritoneal resident cells. All three groups of mice produce comparable titers of specific anti-PC and anti-dextran antibodies. The inability of adult bone marrow-reconstituted BALB/c mice to produce T15 Id+ antibodies is not due to differential VH-gene expression in bone marrow or peritoneum-derived B cells. Thus, the levels of T15 VH in total serum Ig and in anti-PC antibodies are similar in all groups of mice. Furthermore, IEF patterns of T15 VH-associated L chains directly demonstrate differential Vk repertoire expression in bone marrow and peritoneum-derived B cells.     

T cell receptor beta gene sequences in the circular DNA of thymocyte nuclei: direct evidence for intramolecular DNA deletion in V-D-J joining

We have identified circular DNA containing T cell receptor (TCR) beta gene sequences in mouse thymocytes, thereby providing direct evidence for the intramolecular DNA deletion model of V-D-J joining in TCR beta genes. Two types of excision products of V-D-J joining have been identified. Type I, a circular reciprocal recombinant of normal V-D or D-J joining, contains a 7mer-7mer head-to-head structure expected from an excised product of normal V-D or D-J joining. Type II contains a D beta 2-J beta 1 structure on the circular DNA; the recombination event producing this molecule occurs between an upstream J and a downstream D segment, probably leaving the reciprocal 7mer-7mer structure on the chromosome. Some type I molecules seem to represent excision products of secondary joining after formation of the first D-J or V-D-J structure. The recombination mechanism that generates the circular DNA is discussed.     

Somatic evolution of diversity among anti-phosphocholine antibodies induced with Proteus morganii

The variable region sequences of light and heavy chains (VL and VH) were determined for 11 hybridoma antibodies produced in response to the PC moiety on Proteus morganii. These hybridomas were derived from two separate fusions, one obtained from mice early in a secondary response and the other from late in a secondary response. All of these antibodies possessed a cross-reactive idiotype found on anti-PC antibodies in the M603 family, and exhibited preferential specificity for PC in the context of P. morganii. We found that all of the antibodies were derived from a single VH/VL pair. VH was encoded by V1, DFL16.1 and JH1, and VL was encoded by a consensus VK8 gene and JK5. Antibodies differed from each other by somatic point mutations that occurred at a high rate. The mutations in VL were approximately one-third as abundant as those in VH and were randomly distributed throughout the molecule. Mutations in VH were concentrated in CDR 2 and 3 and had a replacement to silent ratio that was three to six times greater than predicted from random accumulation. Based on the sequence data, a single genealogic tree with multiple branches could accommodate all the hybrids from a fusion. We concluded that in both examples the anti-PC response arose by somatic mutation and stepwise selection from a single precursor. Antigen binding studies with these 11 hybridomas and a 12th that had no mutations revealed that the acquisition of preferential specificity for antigen was dependent on somatic mutation of germline genes. Additional binding studies demonstrated that continued selection during clonal expansion was probably antigen driven. An unexpected finding was five independently selected antibodies from one fusion that had identically mutated VH and VL sequences. We suggest that the hypermutation mechanism is not a continuously active process during clonal expansion and that it is regulated, probably during the mid to late phase of the primary response.     

Amino acid sequence of a phosphocholine-binding antibody from an immune defective CBA/N mouse employing the T15 VH region associated with unusual DH, JH, and V kappa segments

Mice expressing the xid gene exhibit an altered immune response to phosphocholine (PC)-conjugated keyhole limpet hemocyanin (KLH). Less than 25% of their anti-PC-KLH response is PC specific, and most of these antibodies lack the normally predominant T15 idiotype. These findings suggested that immune defective mice might employ different variable region genes than normal mice in their anti-PC response. To examine this possibility, we characterized by Southern blot analysis the gene family encoding PC-VH regions and determined the amino acid sequence and fine specificity of binding of a T15-, IgG2, PC-specific hybridoma (1B8E5) produced by fusion of the SP2/O cell line and PC-KLH immune CBA/N spleen cells. Southern blot analysis of DNA from CBA/N mice by using a PC-VH probe (S107 VH) revealed a hybridization pattern virtually identical to that of DNA from normal CBA/J mice, indicating that CBA/N mice do not suffer from a gross deletion of PC-VH genes. Analysis of the 1B8E5 antibody reveals that both the binding specificity and relative affinity of this antibody are different from the anti-PC antibodies of the T15, M167-M511, and M603 families. The complete amino acid sequence of the heavy (H) chain variable region shows that 1B8E5 uses a VH segment identical to the allelic form of T15 (C3) but has a unique D region of three amino acids and use the JH1 joining segment. Both the DH and JH regions are unusual when compared to PC-specific antibodies from normal mice, which have a D region composed of five to eight amino acids and use the JH1 joining segment. The amino terminal sequence of the 1B8E5 light (L) chain demonstrates that this anti-PC antibody carries a Vk3 subgroup L chain. Chains from this subgroup have not previously been found in association with PC-binding antibodies. Thus, the Vk, DH, and JH segments expressed in 1B8E5 make this hybridoma unique in terms of the anti-PC antibodies studied to date, and suggests that additional PC-specific antibodies exist in inbred mice that employ "unusual" V gene segments.     

The amino acid residues at the VH-D-JH junctions affect the affinity of anti-p-azophenylarsonate antibodies

Murine A/J anti-p-azophenylarsonate (Ars) antibodies sharing a predominant idiotype are encoded by a single combination of germ-line V region gene segments. The dominance of this idiotype among secondary immune response anti-Ars antibodies has been explained by the Ag-driven selection of favorable somatic mutants of this gene segment combination, associated with an intrinsic Ars-affinity of the germ-line V region higher than that of other possible combinations. To determine the effect of junctional diversity upon affinity for Ag, independently of somatic mutation, we determined the V region sequences and affinity for Ars of five primary response antibodies. These antibodies share identical unmutated V regions but differ only at the D gene junctions. Among the five antibodies, Ars-affinity differed up to 10-fold depending upon the identity of the amino acid residues at the VH-D and the D-JH junctions. The combination of junctional residues observed in two primary response antibodies with relatively low Ars-affinity has not been observed among secondary response antibodies. Thus the identity of junctional residues resulting from gene rearrangement prior to antigen stimulation must be taken into account in hypotheses which account for idiotype dominance by selection on the basis of affinity.     

B-1a, B-1b and B-2 B cells display unique VHDJH repertoires formed at different stages of ontogeny and under different selection pressures.

Analyses of VHDJH rearrangements isolated from murine peritoneal B-1a cells (CD5+, IgMhi, B220lo), peritoneal B-1b cells (CD5-, IgMhi, B220lo), and conventional splenic B cells provide evidence that a unique repertoire of VH regions is displayed by each of these B-cell subsets. The B-1a subset is characterized by a low N-region diversity, by a high frequency of sequence homologies in the VH-D and D-JH junctions, and by a limited exonuclease nibbling of the terminals of the joining gene segments. Through expansion in ageing mice, B-1a clones with these properties are favoured. B-1b cells are similar to conventional B-2 cells with respect to N-region diversity, but are unique in terms of D gene expression. Thus, while most murine pre-B and B cells preferentially use DSP and DFL gene segments in a given reading frame (RF1), B-1b cells frequently express D genes in another reading frame (RF2). Together, these findings provide structural evidence for a model where B-1a, B-1b and B-2 cells are produced by separate progenitors that are active at different stages of ontogeny.     

Analysis of variable region genes encoding a human anti-DNA antibody of normal origin. Implications for the molecular basis of human autoimmune responses

In order to investigate the genetic basis for natural anti-DNA immune responses, we isolated and sequenced the variable gene elements (VH and VL) encoding an anti-DNA antibody expressed by a human hybridoma of normal origin (Kim4.6) and compared these sequences with those reported for four other human anti-DNA antibodies. The Kim4.6 antibody leader and VH segments were identical in nucleotide sequence with the VH1.9III germ-line VH3 gene, and the Kim4.6VL segment showed 98% nucleotide sequence identity with a V lambda I subgroup gene expressed in a Burkitt's lymphoma. Comparative analysis of Kim4.6 and other human hybridoma anti-DNA antibodies indicated that anti-DNA immune responses are diverse in terms of VH and VL gene utilization but may exhibit a bias toward rearrangement of VH genes that are over-represented in the fetal pre-B cell repertoire. Moreover, Kim4.6 and three of four other sequenced human anti-DNA antibodies appear to use a germ-line diversity gene, DXP'1, which may represent a counterpart of the DFL16.1 segment utilized in murine responses to the hapten nitrophenyl. Taken together, our findings indicate that anti-DNA immune responses can be encoded by nonmutated VH genes and that the elements and molecular mechanisms which engender this response are essentially the same among natural and lupus-associated anti-DNA antibodies. Our data also suggest that natural autoimmune responses originate early in B cell ontogeny as is consistent with the hypothesis that autoreactivity plays a major role in shaping the normal immune repertoire.     

Restricted V-(D)-J junctional regions in the T cell response to lambda-repressor. Identification of residues critical for antigen recognition.

The T cell response to lambda-repressor is directed to a 15 amino acid peptide (P12-26) of the protein in A/J mice. Previous studies have demonstrated a preferential use of V alpha 2 and V beta 1 amongst the T cell hybridomas specific for P12-26 in the context of I-Ek. By using the polymerase chain reaction, the sequences of a panel of the T cells using V alpha 2 and V beta 1 were determined. A highly conserved alpha-chain V-J junctional sequence was found in six of the eight T cell hybrids. This consensus alpha-chain VJ sequence may be combined with different members of V alpha 2, indicating a more restricted selection on the junctional region than on the V element in these T cells. In contrast, greater diversities were found on the V-D-J region of beta-chains despite the same V beta 1 and J beta 2.1 were used. However, a highly conserved glutamic acid residue was found at the same position of beta-chains where a similar conservation was identified in cytochrome c-specific T cells. The correlation of the TCR sequence with the fine specificities of these T cells suggests that a single amino acid deletion in the V alpha-J alpha region may reduce the P12-26 response and abolish the recognition of an altered peptide [Phe22] P12-26. In addition, three amino acid difference in the V-D-J region of the beta-chain also determine the P12-26 reactivity. Thus the V(D)J junctional regions of both alpha- and beta-chains may be critical for the recognition of the peptide Ag presented by the specific MHC molecule.