Detection of a de novo interstitial 2q microdeletion by CGH microarray analysis in a patient with limb malformations, microcephaly and mental retardation.

We describe the cytogenetic diagnosis using BAC- and oligonucleotide microarrays of a 16-year-old Laotian-American female, who first presented at 2 1/2 years of age with microcephaly, developmental retardation, and skeletal abnormalities of the upper limb including mild syndactyly of the second and third and the third and fourth fingers, short middle phalanges and clinodactyly of the fifth digit at the distal interphalangel joint on both hands, and symphalangism of the metacarpal-phalangeal joints of the second and fifth digits bilaterally. Her lower limbs displayed symphalangism of the metatarsal-phalangeal joint of the second, third, and fourth digits on both feet, with fusion of the middle and distal phalanges of the second and fifth digits and hallux valgus bilaterally. G-banded chromosomal study at age 4 was normal. However, comparative genomic hybridization at age 15 with the Spectral Genomics 1 Mb Hu BAC array platform indicated a microdeletion involving two BAC clones, RP11-451F14 --> RP11-12N7 at 2q31.1. The maximal deletion on initial analysis comprised the HOXD cluster, which is implicated in limb development. Fluorescence in situ hybridization (FISH) using the RP11-451F14 probe confirmed the deletion. Both parents were negative for the deletion. Additional FISH using BAC RP11-387A1, covering the HOXD cluster, limited the maximal deletion to approximately 2.518 Mb, and excluded involvement of the HOXD cluster. The Agilent 44K and 244K platforms demonstrated a deletion of approximately 2,011,000 bp, which did not include the HOXD cluster. The malformations in our patient may be caused by deletion of a regulatory element far upstream of the HOXD cluster.

A novel mechanism for B cell repertoire maturation based on response by B cell precursors to pre-B receptor assembly.

The expression of different sets of immunoglobulin specificities by fetal and adult B lymphocytes is a long-standing puzzle in immunology. Recently it has become clear that production of immunoglobulin mu heavy chain and subsequent assembly with a surrogate light chain to form the pre-B cell receptor complex is critical for development of B cells. Here we show that instead of promoting pre-B cell progression as in adult bone marrow, this complex inhibits pre-B cell growth in fetal liver. Curiously, we identify a fetal-associated VH11 mu heavy chain that allows continued pre-B proliferation in fetal liver. Interestingly, this heavy chain does not associate efficiently with a surrogate light chain, providing a previously unrecognized mechanism for skewing the expression of distinctive VH genes toward fetal through early neonatal life.

Human immunoglobulin transgenes undergo rearrangement, somatic mutation and class switching in mice that lack endogenous IgM.

We have generated transgenic mice that contain human-sequence Ig miniloci and, because they are also homozygous for a targeted disruption of their endogenous heavy chain genes, must rely on the transgene sequences for B cell receptor expression. Although the human transgenes contain only a fraction of the intact human heavy chain locus, these defined sequences are able to at least partially restore the humoral immune system in the mouse. B cells expressing human heavy chains develop in the bone marrow, populate peripheral lymphoid tissue and respond specifically to antigen. Furthermore, the heavy chain transgenes contain both human mu and gamma 1 coding exons as well as the respective mu and gamma 1 switch regions. The sequences included within the transgene are sufficient to direct class switch recombination. Transgene sequences are also sufficient to direct somatic mutation of the class-switched heavy chain genes. These observations define the upper limit of the cis-acting sequences necessary to direct heavy chain class switching and somatic mutation.

Distinctive developmental origins and specificities of murine CD5+ B cells.

CD5+ B cells constitute a small fraction of cells in the spleen of adult mice that exhibit numerous features serving to distinguish them from the bulk of IgD++CD5- "conventional" B cells. In this review we focus on two major questions relating to this population: 1) the relationship of CD5+ B cells to other B cells; and 2) the distinctive enrichment of particular autoreactive specificities in this subset. The nature of their origins is clarified by a thorough analysis of intermediate stages of early B-cell development in both fetal and adult tissues. The reactivity to bromelain-treated mouse red blood cells serves as a prototype system for the investigation of biased specificities in CD5+ B cells. These lines of investigation lead us to propose that CD5+ B cells in the adult are the remnant of a distinct fetal B-cell differentiation pathway wherein selection of cells from this fetal/neonatal population into the adult long-lived pool results in the over-expression of certain germline-encoded autoreactivities.

A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins.

We have generated transgenic mice that express a diverse repertoire of human sequence immunoglobulins. The expression of this repertoire is directed by light and heavy chain minilocus transgenes comprised of human protein coding sequences in an unrearranged, germ-line configuration. In this paper we describe the construction of these miniloci and the composition of the CDR3 repertoire generated by the transgenic mice. The largest transgene discussed is a heavy chain minilocus that includes human mu and gamma 1 coding sequences together with their respective switch regions. It consists of a single 61 kb DNA fragment propagated in a bacterial plasmid vector. Both human heavy chain classes are expressed in animals that carry the transgene. In light chain transgenic animals the unrearranged minilocus sequences recombine to form VJ joints that use all five human J kappa segments, resulting in a diversity of human-like CDR3 regions. Similarly, in heavy chain transgenics the inserted sequences undergo VDJ joining complete with N region addition to generate a human-like VH CDR3 repertoire. All six human JH segments and at least eight of the ten transgene encoded human D segments are expressed. The transgenic animals described in this paper represent a potential source of human sequence antibodies for in vivo therapeutic applications.

Variable regions of antibodies to synthetic polypeptides--III. Antibodies arising in response to administration of anti-idiotope.

Antibodies elicited by the synthetic polypeptide antigen (T,G)-A-L are directed against two distinct epitopes. The majority of antibodies bind to a GT containing epitope and bear an idiotope defined by monoclonal antibodies I7 and I9. In this study, we have examined the effect of in vivo administration of the I7 and I9 antibodies to mice. Administration of anti-idiotope elicits anti-(T,G)-A-L antibodies in all strains of mice tested, including genetic non-responders to (T,G)-A-L. These antibodies bind to GT and express the idiotope. Additionally, idiotope expressing antibodies that fail to bind to antigen are also produced. Monoclonal anti-(anti-idiotope) antibodies were made. One antibody bound to (T,G)-A-L, the other did not. Sequence analysis was performed and the V-regions of (T,G)-A-L binding antibodies were compared to those of the antibody that failed to bind antigen. Both sets of antibodies are derived from the same germline V-genes as the anti-(T,G)-A-L antibodies. These results have implications for understanding the nature of network regulation of the immune system and for those attempting idiotypic vaccination.

Influence of a V kappa 8 L chain transgene on endogenous rearrangements and the immune response to the HA(Sb) determinant on influenza virus.

A rearranged murine V kappa 8/J kappa 5 L chain gene that codes for the L chain of most antibodies generated in the primary response of BALB/c mice to the antigenic site, Sb, of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8) has been cloned. Three transgenic lines were generated by microinjecting the gene. Lines Ga and L each contain a single copy of the transgene whereas line Gb contains three complete copies. Mice of the Ga lineage showed increased V kappa 8-specific mRNA levels only in spleen, but not in nonlymphoid organs and therefore displayed apparently normal lymphoid-specific regulation of the Ig transgene. B cell hybridomas generated from these mice were analyzed for rearrangements of endogenous V kappa genes. Greater than 90% of the C kappa alleles were retained in germ-line configuration in the Ga line, compared with only 0 to 18% in the L line. Thus, a wide variation in the frequency of endogenous rearrangements is seen among mice of different lineages using the same transgene construct. None of more than 150 hybridomas derived from LPS-stimulated splenic B cells of Ga mice exhibited HA-binding activity although they expressed the transgene and, in most cases, excluded endogenous V kappa rearrangements. In contrast, a large fraction of hybridomas isolated after primary immunization with PR8 were HA(Sb)-specific. This indicated that the transgene was functional but formed HA-specific antibodies with a more restricted set of H chains than previously hypothesized. The primary anti-HA response to immunization with PR8 was diminished in all lines compared with normal mice except for a slightly accelerated but transient burst of anti-HA antibody formation in two out of three lines (Ga and Gb). This early response in G lineage mice was largely specific for HA(Sb) and thus appeared to be composed of transgene-expressing antibodies. No differences in serum titers were observed in the secondary anti-HA responses to booster inoculation with PR8 between transgenic and normal mice.

The role of clonal selection in the pathogenesis of an autoreactive human B cell lymphoma.

To study the association of autoimmunity and human B cell neoplasia, we have established a model of a B cell lymphoma which expresses a pathogenic autoantibody of defined specificity. The Ig VH gene expressed in this neoplasm was analyzed longitudinally using clinical specimens taken from the splenic lymphoma (S) at diagnosis and from lymph node relapses 3 and 4 yr later (N3 and N4). Southern analysis and oligonucleotide hybridization experiments demonstrated that clonally related predominant and minor tumor cell populations were present in S at diagnosis, and that the minor population became the predominant population in the relapse specimens, N3 and N4. Although the Ig specificity and idiotype were the same at diagnosis and at both relapses, analysis of the expressed VH gene sequences showed 14 base changes between S and N3, and 2 further changes at N4. Little sequence heterogeneity was observed at each sampling time, indicating that the ongoing mutation frequency was low. The relevant germline precursor VH gene was determined from autologous germline DNA and compared to the expressed genes. Based on the pattern of shared and unshared mutations, we were able to establish the genealogic relationship of the germline VH gene and the expressed clonotypes of S, N3 and N4. Taken together, the findings from Southern blotting, oligonucleotide hybridization, and sequence analysis permit us to describe a molecular aspect of tumor progression, "clonotypic shift", wherein subpopulations of the malignant clone, marked by different V gene clonotypes, emerge and predominate at different time points in the evolution of the lymphoma. Furthermore, the sequential and nonrandom pattern of the VH mutations, correlated with the observed conservation of autospecificity and idiotype, implies that clonal selection may have influenced the pathogenesis of the lymphoma.

Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow.

We have resolved B220+ IgM- B-lineage cells in mouse bone marrow into four fractions based on differential cell surface expression of determinants recognized by S7 (leukosialin, CD43), BP-1, and 30F1 (heat stable antigen). Functional differences among these fractions can be correlated with Ig gene rearrangement status. The largest fraction, lacking S7, consists of pre-B cells whereas the others, expressing S7, include B lineage cells before pre-B. These S7+ fractions, provisionally termed Fr. A, Fr. B, and Fr. C, can differentiate in a stromal layer culture system. Phenotypic alteration during such culture suggests an ordering of these stages from Fr. A to Fr. B to Fr. C and thence to S7- pre-B cells. Using polymerase chain reaction amplification with pairs of oligonucleotide primers for regions 5' of JH1, DFL16.1, and Jk1, we find that the Ig genes of Fr. A are in germline configuration, whereas Fr. B and C are pro-B cell stages with increasing D-J rearrangement, but no V-D-J. Finally, functional analysis demonstrates that the proliferative response to IL-7, an early B lineage growth factor, is restricted to S7+ stages and, furthermore, that an additional, cell contact-mediated signal is essential for survival of Fr. A.

Natural autoantibodies to thymocytes: origin, VH genes, fine specificities, and the role of Thy-1 glycoprotein.

15 SM/J mouse hybridoma antibodies that show antithymocyte autoantibody (ATA) activity by immunofluorescence staining were studied. Half of these antibodies react with determinants whose expression is associated with Thy-1, as shown by blocking experiments with anti-Thy-1 and loss of reactivity with Thy-1- mutant cell lines. The Thy-1 dependence of three of these ATA is further confirmed by their reexpression on a Thy-1 gene transfectant. However, the remaining antibodies exhibited binding that showed little or no dependence on Thy-1. Furthermore, we find that most ATA derives from the Ly-1 B subpopulation, as demonstrated by lipopolysaccharide-induced ATA secretion in vitro and by comparison of ATA hybridoma frequencies. VH region gene sequence data of 14 monoclonal ATA from Ly-1 B cell-derived hybridomas reveal the utilization of nine VH genes belonging to four different VH families (J558, 3609, Q52, and Vgam3.8). While we find that two of these hybridomas arose from a clonal expansion, we also find four examples of a 3609 family VH gene utilized in clonally independent lines showing similar specificity. Yet another example of identical VH gene usage by clonally unrelated cells is found in two J558 ATA of a distinct fine specificity. These data suggest that the enrichment of ATA B cells in the Ly-1 B subset is primarily due to repeated independent recruitment of B cells by antigen resulting in the expression of a restricted set of VH genes.

Rearrangement and selection of VH11 in the Ly-1 B cell lineage.

One of the predominant VH genes utilized to encode the anti-BrMRBC specificity is a member of the small VH11 family rearranged to JH1. Using the polymerase chain reaction (PCR) we have determined that the frequency of B cells with a VH11 rearrangement is 10-20 times higher in Ly-1 B than in Ly-1- "conventional" B cells regardless of location (spleen or peritoneal cavity). Conventional B cells rearrange this gene at comparable levels in pre-B cells and in mature B cells utilizing all JH gene segments. In contrast, the increased levels of VH11 rearrangement in Ly-1 B are restricted to JH1 (and some JH2) and therefore appear to be the result of selection. Furthermore, most peritoneal Ly-1 B cells with VH11 rearrangements fall in a fraction stained by anti-BrMRBC antibody, likely bearing multivalent natural (likely self) antigen constitutively bound to their surface Ig receptors. Thus, we suggest that autoantigens are largely responsible for the accumulation of autoantibody specificities in the Ly-1 B cell lineage with time, whereas they do not exert this effect in the conventional B cells.

Relationship of variable region genes expressed by a human B cell lymphoma secreting pathologic anti-Pr2 erythrocyte autoantibodies.

To study the biology of cold agglutinin disease we previously established EBV-transformed B cell clones isolated from a patient with splenic lymphoma of an early plasmacytic cell type and immune hemolysis due to an anti-Pr2 cold agglutinin. These clones had an aberrant chromosomal marker identical to the patient's B cell lymphoma and each secreted IgMk anti-Pr2 similar to the pathologic autoantibody in the serum of the patient. In this study, we have further investigated the Pr2-specific autoimmune response through nucleotide sequencing of VH and VL region genes. We have shown that the seven clones share the same VDJ/VJ gene segments and junctional elements confirming their clonal origin. The VH sequences were 88% homologous to a VHI germline gene while the VL sequences were 97% homologous to a VkIII germline gene. Only 4 somatic mutations (3 silent and 1 conservative) were found in greater than 5,000 bp sequenced, suggesting that a low mutation rate existed. Based on a tumor mass of 10(12) cells and a minimum of 40 divisions, we estimated the somatic mutation rate to be 4.45 x 10(-5) m/bp/d. This somatic mutation rate is similar to those estimated for acute lymphocytic leukemia (pre-B cell) and chronic lymphocytic leukemia (intermediate B cell), but significantly lower than the mutation frequency in follicular lymphomas (activated B cell). We propose that the difference in somatic mutation frequency of a B cell tumor may be related to the stage of B cell differentiation. In addition, the low mutation frequency observed in the Pr2-specific B cell tumor may also reflect, in part, selection by autoantigen to conserve sIg structure and specificity.

A single VH gene is utilized predominantly in anti-BrMRBC hybridomas derived from purified Ly-1 B cells. Definition of the VH11 family.

By establishing hybridomas from two distinct surface IgM+ splenic B cell populations, Ly-1 B cells and "conventional" (Ly-1-) B cells, we found that the Ly-1 B population includes a 30 to 70 times higher frequency (1 to 2%) of cells with specificity for bromelain treated autologous red blood cells (anti-BrMRBC) when compared with conventional B cells (0.03%). We cloned and sequenced the V genes encoding anti-BrMRBC antibody from two hybridomas made with Ly-1 B cells sorted from the spleen of SM/J mice. The VH sequence (for both) is identical with the previously reported sequence associated with this specificity and belongs to a new VH gene family. This gene family, defined here as VH11, has only two members and is the predominant VH rearranged in a collection of Ly-1 B derived anti-BrMRBC hybridomas, always in association with a single VL gene (a member of the V kappa 9 family). Furthermore, analysis of hybridomas made with Ly-1 B cells sorted from the peritoneum reveals a yet higher increased frequency of VH11-encoded anti-BrMRBC specificity (30%). This variation in frequency of anti-BrMRBC in the Ly-1 population depending on location, together with the repeated association of VH11 with a particular V kappa gene suggest that antigen driven selection is (at least in part) responsible for the biased V gene expression seen in this population. Furthermore, a mechanism that might contribute to biased expression, preferential rearrangement due to close proximity to J (as seen in pre-B lines), is excluded by localization of VH11 5' to several of the more J-proximal families (Q52, 7183).

Variable regions of antibodies to synthetic polypeptides. II. Analysis of variable region genes encoding antibodies specific for (T,G)-A--L.

Monoclonal antibodies specific for the synthetic polypeptide antigen (T,G)-A--L have been produced in two strains of mice, C57BL/10 and C3H.SW. The genes encoding the variable (V) regions of these antibodies have been studied by using the DNA hybridization technique of Southern, as well as by gene cloning and sequencing. Hybridization of DNA from 14 different cell lines with a kappa-chain probe revealed that the different cell lines used one of two different gene rearrangements to encode the recombined V region gene. There was a perfect correlation between light chain rearrangement, idiotype expression, and fine specificity. Hybridization analyses of the heavy chain revealed a more complex pattern. Seven hybridomas had the rearranged heavy chain V region genes on a 4.4 kb EcoRI restriction fragment. Others were found on restriction fragments that differed in length by several hundred base pairs. The recombined heavy chain V region genes were cloned from three different hybridoma cell lines secreting anti-(T,G)-A--L antibodies, all of which express the same idiotype and fine specificity pattern. Restriction mapping and sequencing indicate that all three utilize the same V gene, identified as the 186-2 germline gene. However, different D and J genes are used to encode each of the antibodies. In contrast to the results seen in other antigen systems, heavy chain D and J genes do not have a major influence on idiotype expression and fine specificity of antibodies to the synthetic polypeptide (T,G)-A--L.

Antibodies against human lymphokines: I. Methods for induction of antibodies capable of neutralizing stable (alpha) and unstable (beta) lymphotoxins released in vitro by activated human lymphocytes.

Various methods were employed to induce antibodies in rabbits that were capable of neutralizing different families of lymphotoxins (LT). Both stable (alpha-LT) and unstable (beta-LT) molecules, released by activated human lymphocytes in vitro, were neutralized. The different LT families were first separated into their respective groups by physical-chemical methods. Immunization with small quantities of antigen yielded a high percentage of responder animals. Techniques were developed for eliciting alpha-LT antibodies using as little as 2--3 ml of a cell-free supernatant. The situation was more difficult, however, when the unstable beta-LT molecules were employed as antigens. We found that because of the low concentration and lability of beta-LT in supernatants, the immunizing dose had to be: a) handled rapidly, b) larger than that used with the alpha-LT, and c) injected at closer intervals and over a longer immunization protocol. Physical-chemical studies supperted the concept that the LT-neutralizing activity in the immune serum was immunoglobulin.