Faculty Bibliography

The mechanism(s) responsible for the ontogenic patterns of acquisition of the antibody repertoire is unknown. The immune response to Haemophilus influenzae type b (Hib) capsular polysaccharide provides an excellent model system in which to examine the ontogeny of immunoglobulin variable region expression. A panel of hybridomas secreting human antibodies specific for Hib capsular polysaccharide was developed using peripheral blood lymphocytes from donors immunized with Hib vaccines. Nucleotide sequence analysis of the heavy chain V regions expressed by four of these hybridomas suggests selective use of members of the VHIII gene family in combination with different D and J segments. The nucleotide sequences were highly homologous to two candidate germline gene sequences. Others have reported that these particular germline sequences are expressed in fetal liver, suggesting that the inability of young children to produce antibody to the Hib capsular polysaccharide is not due to failure to express these VH regions early in ontogeny

Somatic hypermutation focused to the rearranged V(D)J segment of the immunoglobulin (Ig) loci contributes substantially to antibody gene diversification. However, neither the precise B cell subset subject to hypermutation nor the molecular mechanism(s) involved is known. One model proposes that Ig segments may be uniquely susceptible to DNA nicking and subsequent error-prone repair during a specific B cell developmental stage. We describe an SV40-based shuttle vector system for testing such a model. Plasmids containing two distinct Ig segments juxtaposed to the supF marker gene have been passaged through cell lines representing intermediate stages of B cell development, rescued and screened for marker gene mutations. To date we have not demonstrated enhanced supF mutation in any cell line examined, irrespective of the adjacent Ig segment. Thus, these cell lines exhibit normal DNA repair mechanisms and no evidence of increased endonuclease activity on the Ig segments tested. The feasibility of this system will allow similar experiments using other Ig target sequences exposed to a broader range of B cells

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

Human antibody to the Haemophilus influenzae capsular polysaccharide (Hib CP) is restricted in diversity in the individual and the population with a limited number of variable region genes encoding antibody. Antibody to the Hib CP shows restricted isoelectric focusing gel patterns and light chain usage with frequent restriction to use of only kappa light chains. Shared cross-reactive idiotypes are expressed on antibody. The heavy chain of antibody to the Hib CP is predominantly encoded by two members of the VH3 family--LSG 6.1/M85-like and VH26/30P1-like. In VH the CDR1, based on complete identity in LSG 6.1/M85-like antibodies, CDR2, based on the suggestion of mutation in this region, and CDR3, based on conserved CDR3 usage in unrelated individuals, may be important for antigen binding. Six or more different VL gene families encode antibody. The predominant antibody of the majority of individuals uses the A2-V kappa II gene in germline or near germline configuration, which encodes an idiotype designated HibId-1. Antibody can also be encoded by V kappa I, non-A2 V kappa II, V kappa III, V kappa IV, V lambda II, and V lambda VII genes. Although different VL genes can be used, unrelated individuals appear to use the same V kappa III (A27), V lambda II (V lambda 2.1 and V lambda VII (4A) genes. The VL diversity accounts for differences in fine binding specificity, with A2-V kappa II genes not encoding E. coli K100 CP cross-reactive antibodies and V lambda VII genes and some of the non-A2 V kappa genes encoding cross-reactive antibodies. The arginine in CDR3 of both antibody kappa and lambda light chains and the asparagine in CDR2 of VL sequences and in CDR1 of LSG6.1-M85 VH sequences of antibody appear to be important residues for antigen binding. A relatively limited degree of somatic mutation has occurred in the non-A2 VL genes, V lambda VII, and the VH genes. Further studies comparing the polymorphism of germline V genes to antibody-encoding V genes are needed to clarify this issue. Research comparing this repertoire to repertoires directed to other bacterial CP and to self antigens and defining structure-antigen binding relationships is in progress

Deregulation of c-myc oncogene secondary to chromosomal translocation appears to play an essential role in the genesis of both endemic (African) Burkitt's lymphoma (eBL) and sporadic Burkitt's lymphoma (sBL). In most eBL, mutations in or near exon 1 disrupt normal c-myc regulatory sites. We examined c-myc sequences from a patient with sBL and two patients with eBL to determine (1) whether mutation is ongoing as the tumor clone expands, (2) the nature of mutations in the protein-coding exons 2 and 3, and (3) the extent of c-myc hypermutation in the two clinical forms of BL. Using the polymerase chain reaction (PCR), we amplified segments of c-myc from bulk tumor samples, cloned the products into plasmid vectors, and sequenced multiple subclones of each segment. The mutation frequencies in the control (remission bone marrow) and sBL tumor subclones were 0.65 x 10(-4) and 3.0 x 10(-4) (mutations/base), respectively (P greater than .25). Subclones from the two eBLs exhibited mutation frequencies of 20 x 10(-4) and 16 x 10(-4), respectively (P less than .001 v control). In addition to the consensus mutations seen in one eBL, a random pattern of unshared mutations was observed throughout c-myc in both samples, demonstrating that mutations may be introduced in a stepwise fashion. We noted a clear excess of transitions over transversions (30:9), which is qualitatively similar to the pattern observed in diverse examples of eukaryotic gene mutation. These data demonstrate that c-myc hypermutation is an ongoing process as the eBL tumor clone expands, is qualitatively different from immunoglobulin gene hypermutation, and is not a universal feature of BL, perhaps reflecting the nature of the translocation or the stage of tumor cell maturation

The mechanisms that govern the content of the human antibody repertoire are poorly understood. To investigate the antibody response to a clinically relevant Ag, we have produced heterohybridomas secreting human antibodies directed against the capsular polysaccharide of Haemophilus influenzae type b (Hib PS). Immune lymphocytes were harvested 7 days after immunization with either of two vaccine formulations, a plain polysaccharide vaccine (Hib PS) or a polysaccharide-protein conjugate of Hib PS and diphtheria toxoid (Hib PS-D). H chain V region gene nucleic acid sequences were determined for five stable hybridomas. All use members of the VHIII gene family and are 83% to 98% homologous to two candidate germ-line sequences. A variety of D and JH segments are used. Thus the Ig H chain repertoire appears to be restricted to a limited group of VHIII family members. The previously reported expression of homologous sequences in the human fetal repertoire suggests that the inability of young children to respond to this Ag is not caused by deficiencies of these important elements early in development. The restricted use of VHIII gene segments suggests that this gene family plays a pivotal role in the immune response to this important childhood pathogen

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-

Somatic hypermutation of rearranged Ig V region gene plays a major role in generating antibody diversity. Recently, V mutation has been established as a major mechanism of tumor escape from anti-Id immunotherapy. We cloned and sequenced the expressed Ig H and L chain V regions from a case of B acute lymphoblastic leukemia in order to evaluate B cell stages associated with V region mutation, and to determine which tumors would be better suited to Id directed immunotherapy. A consensus VH and V lambda sequence representing tumor at diagnosis was obtained by conventional cDNA cloning in lambda gt10 from a heterohybridoma. Primers which flanked both V regions were used in a modified polymerase chain reaction to generate multiple independent sequences from tumor cells harvested at relapse. In order to exclude mutations due to infidelity of the amplification procedure, single cDNA templates of known sequence were also amplified. The polymerase chain reaction proved to be an effective procedure to obtain multiple clones, but replication in M13 was associated with a low rate of base misincorporation. The results indicate that there is no evidence for biologically significant ongoing mutation in this t(8;14) B cell tumor when comparing sequences at diagnosis and relapse. Thus, V somatic mutation may be restricted to a discrete B cell stage whose malignant counterpart is follicular lymphoma

CTL are thought to play a role in the elimination of transformed cells in vivo. The effectiveness of such CTL is in part dependent on recognition of tumor specific antigens. Among the best characterized tumor-specific antigens are the unique or idiotypic determinants on the Ig of B cell lymphomas. Here we describe the generation and properties of human CTL specific for the idiotype on autologous B cell tumors. These cells are CD3+,CD4-,CD8- and express the delta chain of the TCR. Such cells may prove useful in tumor-specific adoptive therapy