Caroline J. Chapman

Insight into the origin and clonal history of B‐cell tumors as revealed by analysis of immunoglobulin variable region genes

Summary: Recombination of VH DH and JH genes is a unique first step in normal B‐cell development. Subsequent differentiation to a mature plasma cell is accompanied by further events in the Ig genes, including VI‐Jt joining, somatic hypermutation and isotype switching. Chromosomal changes leading to B‐cell tumors can occur at many points in this sequence, and may be partly a consequence of the genetic mobility and mutability permitted in order to generate a diverse antibody repertoire, V genes of neo‐plastic B cells may reflect the point of maturation reached by the B cell of origin, prior to transformation, Analysis of tumors therefore provides useful information on V‐gene patterns in normal B cells, and may add another dimension to classification of B‐cell tumors. Transformation ma)’ also preserve cell populations normally destined to die by apoptosis. Tumor cells arrested in the sire where somatic hypermutation and isotype switch are occurring can still be subject to these processes, and could be influenced by persisting antigen. However, mutation is silenced at the point of exit lo the periphery, leading lo fixed mutational patterns in tumors of mature B cells, V‐gene analysis provides an invaluable tool for understanding the genesis of neoplastic change. It also has a clear clinical relevance in tracking tumor cells, measuring residual disease, and finally in offering the opportunity of developing vaccines for treatment.

Insight into Burkitt's Lymphoma from Immunoglobulin Variable Region Gene Analysis

Analysis of usage of V(H) and V(L) genes, and the degree and pattern of somatic mutation, has been used to investigate the cell of origin and clonal history in cases of Burkitts lymphoma (BL). Tumor cell lines and biopsy material from patients with endemic, sporadic and AIDS-associated BL have been compared. V(H) genes were most commonly derived from the V(H)3 (52%) and V(H)4 (39%) families. This shows a similar gene usage of the V(H)3 family to that seen in the normal peripheral blood repertoire (55%), but a biased usage of the V(H)4 family (22% in normal). There was no restriction in V(L) gene usage. This overall distribution was similar in all subsets of BL. In all categories, there was significant somatic mutation in both V(H) and V(L) sequences. There was no evidence for accumulation of mutations in cell lines cultured in vitro indicating that all mutations in BL-derived cell lines have accumulated in vivo. The mean percentage level of mutation +/- standard deviation was greater in endemic BL (V(H) = 7.7 +/- 4.0, V(L) = 5.3 +/- 2.2) and AIDS-associated BL (V(H) = 7.5 +/- 3.6, V(L) = 3.9 +/- 1.9) than in sporadic BL (V(H) = 4.0 +/- 2.5, V(L) = 2.2 +/- 1.2). The pattern of somatic hypermutation was similar in V(H) and V(L) sequences of the different types of BL although the light chain genes were less mutated. Mutational patterns in the V(H) genes did not reveal a conventional role for antigen in selection of tumor cell sequences in 23/25 V(H) genes analysed. In contrast, patterns in V(L) sequences were consistent with a role for antigen in 8/13 sBL +/- eBL cases and 8/17 cases overall. The presence of EBV did not seem to influence the quantity or pattern of somatic mutations. Evidence for intraclonal variation was seen in uncloned cell lines from cases of eBL and AIDS-associated BL and confirmed in biopsy material in some, but not all cases of eBL, sBL and AIDS-associated BL examined. These common features indicate that the B-cells involved in all types of BL are derived from cells that have traversed the germinal centre, and that the somatic mutation mechanism may still be operative following neoplastic transformation. Overall, in 10/30 cases, there was evidence of significant clustering of replacement amino acids, in CDRs, particularly in V(L), indicating that the B-cell of origin is likely to have been selected by antigen.

Dual recognition of lipid A and DNA by human antibodies encoded by the VH4-21 gene: a possible link between infection and lupus.

The VH4-21 (V4-34) gene segment, a member of the VH4 family, is expressed early in B-cell maturation and is utilized by approximately 6% of normal adult B lymphocytes. This prevalence indicates an importance of VH4-21 in the B-cell repertoire. The gene also encodes certain autoantibodies being mandatory for pathological IgM anti-red cell antibodies directed against the I/i antigen, and also capable of encoding anti-DNA antibodies. Recognition of I/i antigen or DNA appears to be via two distinct sites on VH, with I/i binding mediated by sequences in the framework region, and DNA binding correlating with the presence of positively charged amino acids in complementarity-determining region 3. However, these positively charged residues appear to suppress the ability of the framework region to interact with I/i, rendering a single sequence monospecific for I/i or DNA. The IgM anti-DNA antibodies also recognize bacterial lipid A, whereas the anti-I/i antibodies do not, indicating that CDR3 may be involved in binding the negatively charged lipid A. Structural similarities between the DNA backbone and lipid A provide a possible explanation for this cross-reactivity. This dual recognition of bacterial antigen and autoantigen provides a potential link between infection and autoimmunity.

The HPV-activating cellular transcription factor Brn-3a is overexpressed in CIN3 cervical lesions.

The cervical cellular transcription factors Brn-3a and Brn-3b have antagonistic effects on transcription of the human papilloma virus types 16 and 18 E6 and E7 oncogenes, with Brn-3a activating expression and Brn-3b repressing it. We therefore measured expression of Brn-3a and Brn-3b mRNAs in biopsies from 16 women with no detectable cervical abnormality, and in 14 women with cervical intraepithelial neoplasia grade 3 (CIN3) lesions. Although the mean level of Brn-3b expression was similar in both groups, the mean level of Brn-3a expression was over 300-fold higher in the CIN3 samples when compared with normals. Elevated expression of Brn-3a was also detected in 16 histologically normal regions of the cervix adjacent to the CIN3 lesions, indicating that elevation of Brn-3a levels is not confined to the lesion in women with CIN3, and is thus not a consequence of the oncogenic process. The elevated levels of Brn-3a in the CIN3 patient samples, together with the activating effect of Brn-3a on HPV-16 and -18 oncogene expression, suggest that induction of this factor is involved in activating HPV-16 and -18 oncogene expression in the cervix, and hence in the production of cervical cancers induced by HPV.

Structural Analysis of VH4–21 Encoded Human IgM Allo- and Autoantibodies Against Red Blood Cells

We have sequenced the variable heavy chain regions of a number of VH4–21 encoded monoclonal IgM anti‐Rh(D) antibodies produced in response to deliberate immunization. These were compared with the sequences of similarly encoded IgM anti‐I cold agglutinins (CA) derived from patients with lympho‐proliferative diseases. The anti‐Rh(D) antibodies show evidence of clonal expansion and somatic diversification. Even though they are produced in response to an antigenic stimulus, they demonstrate limited hypermutation in the variable heavy chain (VH) segments and there is no evidence of selective pressure acting on the complementarity determining regions (CDRs). The CA demonstrate a higher rate of mutation and yet this results in a lower ratio of replacement to silent mutations (R:S) in the CDRs than seen in the anti‐Rh(D) antibodies. It is not clear whether the different pattern of mutations seen in the CA is related to their auto‐reactivity or their tumour origin. In both groups of antibodies the region encoded by the VH4–21 segment can be found in germline configuration at the amino‐acid level indicating that other V‐gene structures, i. e. light chains or CDRH3s, are crucial to the generation of either specificity. A role of the CDRH3 is indicated by the identification of a motif shared by four CAs and one Rh(D) antibody which also demonstrates CA activity independent of its anti‐Rh(D) specificity. Amongst the anti‐Rh(D) antibodies there seems to be an obligatory combination with VL having closest homology to the DPL16 germline segment indicating this as particularly important in generation anti‐Rh(D) specificity.

Clonally related IgE and IgG4 transcripts in blood lymphocytes of patients with asthma reveal differing patterns of somatic mutation

Isotype switching to IgE contributes to atopic asthma, therefore strategies to divert this process to alternative isotypes could have therapeutic relevance. It is known that patients with allergic disease have serum IgE and IgG4 antibodies with similar specificities, and that cytokines such as IL‐4 mediate switching to both of these isotypes. Availability of variable region gene analysis has allowed us to probe isotype variants at the single‐cell level. An earlier report described identification in a single atopic patient of short transcripts with a common complementarity‐determining region “clonal signature” in combination with Cμ, Cγ4 and Cϵ. We have extended this analysis, and have identified VH‐Cγ4 transcripts with clear clonal relationship to IgE‐derived sequences in blood lymphocytes from three of four patients with atopic asthma. No other IgG subclasses were detected, confirming the link between IgE and IgG4. Full sequences were obtained from each clonally related isotype in all patients, and showed extensive somatic mutation. As previously found for IgE, the IgG4 isotypes had evident intraclonal variation. There were shared mutations between isotypes, but also many differences, indicative of separate cell populations with divergent mutational histories. These findings indicate that, in atopic patients, an individual B cell commonly switches to either IgE or IgG4. Cells producing each isotype then co‐exist in the recirculating pool, and the balance between them may influence the disease process.

Immunogenetic analysis of the heavy chain variable regions of IgE from patients allergic to peanuts.

Peanuts are one of the most allergenic of the foods, and hypersensitivity responses to peanut allergens can be fatal. Although the nature of the antigenic components of peanuts is being defined at the molecular level, there is little information on the induced IgE antibodies, which are central to the allergic reaction. Recognition sites of IgE antibody molecules arise from the variable regions of heavy and light chains (VH and VL). By using nested polymerase chain reactions with specific primers for the available repertoire of VH genes, together with primers in the constant epsilon region, we have amplified VH sequences of IgE from blood lymphocytes of two patients with peanut allergy. After cloning and sequencing the products, we found a predominance of VH1 family use in both patients, which was not found in control IgM-specific primers. The IgE VH sequences were highly somatically mutated, but in only six of 17 cases was there clear evidence for clustering of amino acids indicative of antigen selection. Previous results from patients with allergy to house dust mites have indicated predominance of VH5 use and little evidence for antigen selection. Although results from two patients allergic to peanuts must be regarded as preliminary, they do suggest that the IgE response to peanuts may have a different VH bias, with a similar mutational pattern.

Tracking of the V4–34 (VH4–21) gene in human tonsil reveals clonal isotype switch events and a highly variable degree of somatic hypermutation

The V4–34 (VH4–21) gene has been found to encode certain IgM autoantibodies, and is mandatory for pathological IgM anti‐erythrocyte antibodies of I/i specificity. The gene is also commonly used by normal IgM‐positive B lymphocytes, but its involvement in B cells which have undergone class switching to IgG or IgA is less clear. In order to track V4–34 gene usage and class switching events during a normal immune response, we have probed RNA in a limited area of human tonsil. Results indicate that the V4–34 gene undergoes class switching to IgG or IgA, with the progeny either remaining unmutated or containing large numbers of somatic mutations. Mutational patterns indicate possible ‘hot spots’, and some mutations appear deleterious. At the level of individual B cells, we have tracked a clonal isotype switch event from IgM to IgA, with each retaining close to germ‐line configuration. In addition, we have followed a clonal switch from a mutated IgM to IgG, with no further accumulation of somatic mutations. These data indicate that the V4–34 gene is involved in a maturing immune response, and that the routes to production of IgG or IgA antibodies are various.

Differential Usage of an Autoantibody-associated VH Gene, VH4-21, by Human B-cell Tumors

Selection of immunoglobulin variable region genes for recombination in B cells takes place from among those VH and VL gene segments available in the unrearranged germ line repertoire. In the case of neoplastic B cells, there is apparent deviation in the use of V-genes from that expected on a random basis, both for VH and for VL. Also, the preferred V-genes, and their patterns of mutation, differ among the various categories of B-cell tumor possibly reflecting the distinct origins and clonal histories on the individual tumor cells. This review focuses on a single VH gene, VH4-21, which is a member of the VH4 family, and which appears selectively to encode immunoglobulins with autoantibody activity, particularly anti-red cell antibodies. The pattern of usage of this VH gene by B-cell tumors demonstrates clear asymmetry among different tumor types. Also, the mutations detected in this relatively non-polymorphic gene indicate that antigen, possibly autoantigen, may influence the behavior of the tumor cell.

Pattern of usage of the VH4-21 gene by B lymphocytes in a patient with EBV infection indicates ongoing mutation and class switching.

Following infection with EBV, patients have selectively raised serum levels of immunoglobulins encoded by the VH4-21 gene. In order to follow the detailed pattern of usage of the VH4-21 gene by blood B lymphocytes of a typical patient during infection, EBV lines were established, and transformed B cells were hybridized and cloned. In addition, to widen the genetic analysis, cDNA preparations from the EBV transformants using the gene were also analysed by polymerase chain reaction, cloning and sequencing. The majority (12/15) of the clonally distinct sequences derived from IgM utilized the VH4-21 gene in germ line configuration; however, 3/15 showed replacement mutations. For one of these, a heterogeneous pattern of mutation within the clone indicated ongoing mutation, and one sequence contained a stop codon. Three distinct clones which had rearranged to C gamma were obtained, and all were extensively mutated, with some evidence for a role for antigen in selection. Following resolution of the infection, no VH4-21-encoded products were detectable by this approach. It appears therefore that infection with EBV leads to selective activation, mutation and class switching of the VH4-21 gene, with the unusual feature that B cells harbouring deleterious mutations in the functional gene are able to survive in the circulation.