William L. Carroll

Mouse × human heterohybridomas as fusion partners with human B cell tumors☆

Surface idiotype (Id) of B cell malignancies is an excellent tumor-specific marker. We have, however, recently described heterogeneity of tumor Id in some cases. We therefore sought a way to isolate, reliably and efficiently, different species of idiotype from a potentially heterogeneous population. In this report we demonstrate our success using a series of mouse X human heterohybridomas as fusion partners with human B cell tumors. Three lines (K6H6/B5, K6H9/G12, SBC/H20) demonstrated excellent fusion efficiency with 75%-85% of wells plated containing hybrids. Two cell lines, K6H9/G12 and SBC/H20 had a tendency to secrete a single Ig chain (heavy or light chain), whereas the K6H6/B5 cell line secreted whole immunoglobulin (Ig) in greater than 80% of the hybrids. This line secreted significant amounts of Ig (2.73 micrograms/ml/10(6) cells) and was relatively stable in culture. Since this line has such a high fusion efficiency the products of normal B cells admixed with tumor may be recovered, allowing the opportunity of isolating host anti-tumor antibodies. In order to prove that hybrids were derived from the tumor, Southern blot analysis of rearranged DNA was performed in selected cases. Fusions with this line provide the potential for recovering many different species of idiotype in a mixed population. This will facilitate the production of mouse monoclonal anti-idiotype antibodies against many variants and against different idiotopes.

Somatic Mutation in Human B-Cell Tumors

A widely held view has persisted for many years that malignant cells of human tumors are usually incapable of many of the regular physiologic functions of their normal counterparts. Even if competent to carry out some limited functions, it is frequently considered that they often do so in an aberrant, defective way, such as the secretion of Bence-Jones protein from myeloma cells. This generalization is probably an oversimplification; in fact, malignant cells may display a variety of activities possessed by normal cells that show similar differentiated features. For instance, malignant cells of human chronic myelocytic leukemia in lymphoid blast crisis may rearrange their immunoglobulin genes to produce apparently normal surface immunogiobulin {Korsmeyer et al. 1983). Recently, we have investigated another characteristic of some human lymphoid cancers which is considered to be highly specific for certain stages of B-cell development. This characteristic is the ability to selectively mutate their rearranged immunoglobulin genes. The discovery of somatic mutation in human B-ceil tumors unfolded during the course of studying the interaction of human B-cell neoplasms with anti-idiotype antibodies. Starting from the assumption of uniformity of the cell surface idiotypes within the tumor, anti-idiotype antibodies were used to identify tumor cell populations. We found that idiotypes were not homogeneously expressed by all cells within these tumors, and that idiotypic heterogeneity results from hypermutation of immunoglobulin genes within the cells of the evolving tumor. Since our studies indicate that somatic mutation in tumor cells closely resembles that in normal B cells in several respects, we expect that human lymphoma cells will offer several advantages as a system for investigation of the hypermutation process in immunoglobulin genes.

Hybridoma fusion cell lines contain an aberrant kappa transcript.

The V region sequence of a non-productive kappa transcript from two myeloma fusion partners has been determined. This transcript has an aberrant VJ recombination site resulting in a translation stop site at position 105. It is variably expressed in hybridomas made from all fusion partners derived from the original MOPC-21 tumor. The amount of this transcript may greatly exceed levels of the productive light chain mRNA.

Restricted immunoglobulin VH usage and VDJ combinations in the human response to Haemophilus influenzae type b capsular polysaccharide. Nucleotide sequences of monospecific anti-Haemophilus antibodies and polyspecific antibodies cross-reacting with self antigens.

To examine the human antibody repertoire generated against a biologically significant antigen we have obtained sequences of heavy chain variable region genes (IgVH) from 15 monoclonal antibodies specific for the capsular polysaccharide of Haemophilus influenzae type b (Hib PS). All VH segments are members of the VH3 family and 9 of 15 are members of the smaller VH3b subfamily. Restriction is evident by the shared use of certain VDJ joints in independent hybridomas from different subjects. Two hybridomas generated from the same subject demonstrate identical heavy chain variable region gene sequences but differ in isotype and rearrange alternative light chain variable region genes (IgVL), suggesting that in a normal immune response, a single pre-B cell clone may use different light chain rearrangements and give rise to progeny capable of reacting with antigen. Using a polymerase chain reaction assay optimized to detect base pair differences among VH genes we demonstrate that at least a portion of expressed anti-Hib PS VH genes have undergone somatic mutation. Anti-Hib PS heavy chain genes are homologous to VH segments encoding autoantibodies and two hybridomas secrete anti-Hib PS antibody that cross-reacts with self antigens (double-stranded DNA and single-stranded DNA). Comparison of VH regions of self-reactive and monospecific anti-Hib PS Ab demonstrates no consistent structural feature correlating with fine antigen specificity. These data demonstrate significant restriction in VH usage and VDJ recombination in the anti-Hib PS response and confirm that autoantibodies may be elicited during normal immune responses.

Familial bone marrow monosomy 7. Evidence that the predisposing locus is not on the long arm of chromosome 7.

Loss of expression of a tumor-suppressing gene is an attractive model to explain the cytogenetic and epidemiologic features of cases of myelodysplasia and acute myelogenous leukemia (AML) associated with bone marrow monosomy 7 or partial deletion of the long arm (7q-). We used probes from within the breakpoint region on 7q-chromosomes (7q22-34) that detect restriction fragment length polymorphisms (RFLPs) to investigate three families in which two siblings developed myelodysplasia with monosomy 7. In the first family, probes from the proximal part of this region identified DNA derived from the same maternal chromosome in both leukemias. The RFLPs in these siblings diverged at the more distal J3.11 marker due to a mitotic recombination in one patient, a result that suggested a critical region on 7q proximal to probe J3.11. Detailed RFLP mapping of the implicated region was then performed in two additional unrelated pairs of affected siblings. In these families, DNA derived from different parental chromosome 7s was retained in the leukemic bone marrows of the siblings. We conclude that the familial predisposition to myelodysplasia is not located within a consistently deleted segment on the long arm of chromosome 7. These data provide evidence implicating multiple genetic events in the pathogenesis of myelodysplasia seen in association with bone marrow monosomy 7 or 7q-.

Immunoglobulin light chain variable region gene sequences for human antibodies to Haemophilus influenzae type b capsular polysaccharide are dominated by a limited number of V kappa and V lambda segments and VJ combinations.

The immune repertoire to Haemophilus influenzae type b capsular polysaccharide (Hib PS) appears to be dominated by certain light chain variable region genes (IgVL). In order to examine the molecular basis underlying light chain bias, IgVL genes have been cloned from a panel of heterohybridomas secreting human anti-Hib PS (antibody) (anti-Hib PS Ab). One hybridoma, representative of the predominant serum clonotype of anti-Hib PS Ab in older children and adults following immunization or Hib infection, uses a V kappa II segment identical to the germline gene A2, and a JK3 segment. A second kappa hybridoma uses a member of the V kappa I family and a JK4 segment. Four lambda antibodies, all cross-reactive with the structurally related antigen Escherichia coli K100 PS, use V lambda VII segments which are 96-98% homologous to one another, and may originate from a single germline gene. Two additional lambda antibodies, not K100-cross-reactive, are encoded by members of the V lambda II family. All lambda antibodies use highly homologous J lambda 2 or J lambda 3 segments. The VJ joints of all lambda antibodies and the V kappa II-encoded antibody are notable for the presence of an arginine codon, suggesting an important role in antigen binding. Although more complex than heavy chain variable region gene usage, a significant portion of serum anti-Hib PS Ab is likely to be encoded by a limited number of V kappa and V lambda segments and VJ combinations, which may be selectively expressed during development, or following antigen exposure.

Childhood bone marrow monosomy 7 syndrome: a familial disorder?

Description dune fille decedee a 6 ans 1/2 et de son frere en vie a lâge de 8 ans, tous deux souffrant dun syndrome myeloproliferatif precurseur chez la fille dune leucemie aigue non lymphocytaire, et tous deux porteurs dune monosomie 7 dans 100% des cellules medullaires tandis que le caryotype des lymphocytes peripheriques etait normal. La monosomie 7 medullaire est souvent familiale, ce qui rend souhaitable une surveillance des germains des sujets monosomiques 7 par numeration periodique des globules, dosage de lhemoglobine fœtale et au moindre doute ou avant un don de moelle en vue dune greffe eventuellement, un caryotypage des cellules medullaires

Development of the human antibody repertoire.

Humans are confronted with a vast array of foreign antigens, necessitating the generation of a large number of protective Ig molecules. It is estimated that the immune system is capable of giving rise to lOI4 or more B cell clones, each synthesizing a unique antibody species with a distinct antigen specificity. In recent years, the molecular basis of this remarkable diversity has become increasingly clear. However, much of this information is derived from studies of the immune response of experimental animals to simple antigens, and it is apparent that significant differences exist among higher vertebrates both in the structure of Ig genes and in mechanisms used to generate diversity. This review will summarize advances in the understanding of the molecular basis for antibody diversity with particular attention to the developmental expression of the antibody repertoire. It will also focus on how molecular analysis of the human antibody response to Hib may further our understanding of the process by which humans develop a protective immune response. The human immune response to Hib is an excellent model with which to study the development of the human antibody repertoire. This organism is an important cause of serious bacterial infections, and antibody directed against its capsular polysaccharide protects against invasive infection (1, 2). Similar to the response to some other polysaccharide antigens, antibody to Hib polysaccharide develops relatively late in ontogeny. Thus, children less than 5 y of age demonstrate poorer antibody responses to Hib colonization, to infection, and to vaccination with plain Hib polysaccharide vaccine than older children or adults and therefore are most susceptible to infection (2). Coupling Hib polysaccharide to carrier proteins significantly increases immunogenicity of Hib vaccines (3). In addition to the age-dependent acquisition of anti-Hib polysaccharide antibody, certain ethnic groups and individuals have an increased risk of Hib infection (4-6). It has been hypothesized that both phenom-

Conservative Management of Testicular Endodermal Sinus Tumor in Childhood

Endodermal sinus tumor is the most common testicular neoplasm in childhood. The management of children with this neoplasm remains controversial. We have treated prospectively 5 children with stage I endodermal sinus tumor with limited surgery and no adjuvant therapy. The median patient age at diagnosis was 21 months (range 5 to 24 months). All children underwent an inguinal orchiectomy with high ligation of the spermatic cord. Retroperitoneal node dissection was not performed in any case and no child received adjuvant chemotherapy or radiation therapy. All patients were well without evidence of recurrent disease at a median followup of 46 months (range 19 to 72 months). Because these tumors usually are localized at the time of diagnosis, rarely spread to the retroperitoneal nodes and have a biological marker in most cases, and because good salvage chemotherapy is available for patients with relapse, we believe that nonmetastatic testicular endodermal sinus tumors in children can be managed with radical orchiectomy alone. Retroperitoneal node dissection is not necessary and adjuvant therapy is not indicated if markers return to normal. Further treatment should be reserved for the rare child with relapse.

Diversity of immunoglobulin light chain usage in the human immune response to Haemophilus influenzae type b capsular polysaccharide.

ABSTRACT: The response to the capsular polysaccharide of Haemophilus influenzae type b (Hib PS) has been used to determine the molecular basis of antibody gene diversity in humans. In contrast to the relatively restricted nature of anti-Hib PS heavy-chain variable region gene expression, a variety of light-chain variable region genes may encode this antibody (Ab) response. Light-chain variable region gene usage appears to determine the expression of certain Ab idiotypes and fine antigen specificity. To further define the role of light-chain variable region gene usage in important anti-Hib PS Ab subgroups, we have cloned and sequenced a number of immunoglobulin light-chain variable region genes (IgVL) from human monoclonal IgA anti-Hib PS Ab generated in response to Hib PS-protein conjugate vaccines. Three of these Ab are encoded by unusual variable segments. One κ-Ab is encoded by the “predominant” VkII A2 germline gene but, in contrast to a previously reported A2-encoded IgVL sequence, differs from the A2 germline sequence. The IgVL sequence of a second Ab is the only sequence of a κ-Ab that cross-reacts with the structurally related antigen Escherichia coli K100 polysaccharide reported to date. This IgVL is encoded by a VkIII-segment most closely homologous to the Humhv328/L16 germline gene, whereas previous reports suggested VkIII-encoded anti-Hib PS Ab might be exclusively encoded by the germline gene Humhv325/A27. A λ-Ab, also cross-reactive with K100 polysaccharide, is encoded by a V