Preparing the ground for vaccination against multiple myeloma.
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Animals↗
Biomedical subjects
F K Stevenson
Publications and source records attributed to F K Stevenson.
IgG-secreting lymphoplasmacytoid leukaemia: a B-cell disorder with extensively mutated VH genes undergoing Ig isotype-switching frequently associated with trisomy 12.
We investigated 16 patients with elevated serum monoclonal IgG and a leukaemic B-cell lymphocytic disorder different from multiple myeloma. Their clinical history was that of a non-aggressive disease with dominant splenomegaly and long survival. Whereas abnormal blood and bone marrow cells were predominantly small lymphocytes with a few lymphoplasmacytoid cells, histopathological features included a lymphoplasmacytic infiltrate in eight cases. Most frequently, abnormal blood cells displayed a CD19+CD5-CD23+/- immunophenotype different from that of chronic lymphocytic leukaemia, except in two cases with a CD19+CD5+CD23+ phenotype. Interestingly, a coexistent serum monoclonal IgM and/or surface IgMG+ with identical light chain was identified in 10 patients, whereas in the remaining six patients only IgG expression was determined. VH gene analysis was performed in eight patients to investigate the clonal origins of tumour cells. All cases utilized the VH3 family, with evidence of extensive somatic mutations and intraclonal homogeneity in all cases. VH gene analysis indicated a clonal relationship between cells expressing IgM and IgG, with one case being biclonal. Cytogenetic evaluation showed a high incidence of trisomy 12 (60%) and 13q14 deletion (40%). In conclusion, we have described an unusual subset of low-grade lymphoma with high-serum IgG and frequent lymphoplasmacytoid features in which tumour cells derive from post-follicular memory B cells undergoing isotype switching with some cases arrested at both the IgM and IgG stage and others as IgG-positive cells only.
Adult↗
Recognition of auto- and exoantigens by V4-34 gene encoded antibodies.
The antigenic specificities of 24 V4-34-encoded monoclonal antibodies were compared with the amino acid sequence. The specificities were divided into three categories, red blood cells, B lymphocytes and auto/exoantigens. Six anti-I monoclonal antibodies, with multiple substitutions in their VH region, did not bind B lymphocytes or auto/exoantigens. Reactivity to these two antigens segregated with the 16 anti-i monoclonal antibodies, which were derived from the near germline V4-34 gene. All anti-i monoclonal antibodies bound B lymphocytes, albeit with varying intensities. B-cell binding correlated with basic amino acids in the VH-CDR3. Reactivity to auto/exoantigens was demonstrated only by a subset anti-i monoclonal antibodies and did not correlate with B-lymphocyte or i-antigen binding. These anti-ssDNA reactive monoclonal antibodies had basic amino acids in the VH-CDR3, strongly supporting the suggested role of arginine in DNA binding. However, an arginine-rich CDR3 was not enough to ensure DNA reactivity, since six other anti-i monoclonal antibodies that fulfilled this criteria did not bind ssDNA. Thus it is possible that the anti-DNA reactivity of V4-34-encoded monoclonal antibodies is mediated by the classic antigen-binding groove generated by the CDRs of the heavy/light chains. In contrast, anti-B-cell/i-antigen reactivity is mediated, unconventionally, by the V4-34 protein with a dominant influence of the VH-CDR3.
Amino Acid Sequence↗
Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia.
Despite having several characteristics of naïve B cells, chronic lymphocytic leukemia (CLL) cells have been shown in some cases to have somatically mutated Ig variable region genes, indicating that the cell of origin has passed through the germinal center. A previous study of patients with CLL found an association between lack of somatic mutation and trisomy 12 and, therefore, possibly with a less favorable prognosis. We have sequenced the Ig V(H) genes of the tumor cells of 84 patients with CLL and correlated our findings with clinical features. A total of 38 cases (45.2%) showed >/= 98% sequence homology with the nearest germline V(H) gene; 46 cases (54.8%) showed >2% somatic mutation. Unmutated V(H) genes were significantly associated with V1-69 and D3-3 usage, with atypical morphology; isolated trisomy 12, advanced stage and progressive disease. Survival was significantly worse for patients with unmutated V(H) genes irrespective of stage. Median survival for stage A patients with unmutated V(H) genes was 95 months compared with 293 months for patients whose tumors had mutated V(H) genes (P =.0008). The simplest explanation is that CLL comprises 2 different diseases with different clinical courses. One, arising from a memory B cell, has a benign course, the other, arising from a naïve B cell, is more malignant.
Adult↗
V(H) gene sequences from primary central nervous system lymphomas indicate derivation from highly mutated germinal center B cells with ongoing mutational activity.
Primary central nervous system lymphoma (PCNSL) represents 1% to 3% intracranial tumors. Most PCNSL are located in the brain, and 75% are large B-cell lymphomas. The largest subgroup of these tumors contains cells that resemble centroblasts and has been labelled diffuse centroblastic (polymorphous) lymphoma. To investigate the cell of origin and the clonal history of these tumors, we have analyzed V(H) gene of 5 cases of PCNSL, all confirmed by histological studies to be Epstein-Barr virus (EBV)-negative, high-grade diffuse B-cell lymphomas. The V4-34 gene of the V(H)4 family was used in 4 of 5 cases. All V(H) genes were found to have accumulated very high levels of somatic mutation (14% to 25%). In 3 of 5 cases, intraclonal nucleotide heterogeneity, including codon deletion in some clones in 1 case, was observed, indicating that the V(H) genes were still under the influence of the somatic hypermutation mechanism. Analysis of the distribution of silent and replacement mutations showed evidence for preservation of immunoglobulin structure in all cases. These results suggest that, although there is no evidence for germinal center formation in the brain tissue, PCNSL is derived from a B cell with features associated with location in a germinal center environment.
Aged↗
Manipulation of pathogen-derived genes to influence antigen presentation via DNA vaccines.
To gain insight into the routes of presentation of pathogen sequences via DNA vaccines, we have compared the abilities of sequences encoding fragment C of tetanus toxin (FrC) and influenza A virus nucleoprotein (NP) to induce antibody or cytotoxic T-cell (CTL) responses in vivo. Strong antibody and CTL responses were induced against FrC targeted to the endoplasmic reticulum (ER) and both were reduced by removal of the leader sequence. In contrast, targeting of NP to the ER generated only a modest antibody response, likely due to misfolding in this site. Removal of the leader sequence led to anti-NP antibodies via cross-priming. For NP, induction of CTLs was not influenced by the leader sequence. Exogenous FrC or NP delivered as proteins were unable to induce CTLs. Routes to induction of optimal immune responses via DNA evidently differ according to the nature of the encoded pathogen sequence. Understanding processing pathways for pathogen sequences should assist rational design of DNA vaccines.
Animals↗
V(H) gene analysis of IgM-secreting myeloma indicates an origin from a memory cell undergoing isotype switch events.
IgM-secreting plasma cell tumors are rare variants of typical isotype-switched multiple myeloma with a similar disease outcome. To probe the origin and clonal history of these tumors, we have analyzed V(H) gene sequences in 6 cases. Potentially functional tumor-derived V(H) genes were all derived from V(H)3, with the V(3-7) gene segment being used by 4 of 6. All were somatically mutated, with a mean deviation from germline sequence of 5.2% (range, 3.1% to 7.1%). The distribution of replacement mutations was consistent with antigen selection in 4 of 6 cases, and no intraclonal heterogeneity was observed. Clonally related switched isotype transcripts were sought in 4 cases, and Cgamma transcripts with tumor-derived CDR3 sequence were identified in 2 of 4. These findings indicate that IgM-secreting myelomas are arrested at a postfollicular stage at which somatic mutation has been silenced. Isotype switch variants show the cell of origin to be at the IgM to IgG switch point. These features indicate that the final neoplastic event has occurred at a stage immediately before that of typical isotype-switched myeloma. One possibility is that IgM myeloma involves the previously identified precursor cell of typical myeloma.
Aged↗
Autoantibodies revealed: the role of B cells in autoimmune disease.
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Autoantibodies↗
DNA vaccines against cancer: from genes to therapy.
After an erratic history, there is at last a clear opportunity for mobilizing an immune attack against cancer cells. The new strategies are dependent on the techniques of molecular biology, which are able both to identify potential target tumor antigens at the gene level, and to help to unravel the complexities of immune mechanisms required. Vaccine delivery systems can also be genetic, with DNA vaccines able to act as viral mimics and enter several antigen processing pathways. Rational vaccine designs can be rapidly tested in models and selected for pilot clinical trials. One difficulty faced by tumor antigens is that they may be weak, and therefore fail to engage the immune system. Attaching genes encoding alert signals appears to solve this problem. We have focused initially on idiotypic determinants of B-cell tumors, where the encoding variable region genes can induce protective anti-idiotypic immunity if delivered as a fusion protein with a fragment of Tetanus toxin. This model may have relevance for alternative tumor antigens. A clinical trial of patients with lymphoma is in progress, and wider application may be limited only by the ability to bring patients into clinical remission prior to vaccination.
Animals↗
VH gene sequences from a novel tropical splenic lymphoma reveal a naive B cell as the cell of origin.
The prevalence of malaria and other infections in tropical Africa provides a setting for the emergence of B-cell tumours distinct from that in Western countries. Attempts to draw comparisons with Western lymphomas have led to difficulties, with so-called African chronic lymphocytic leukaemia (CLL) having a different pattern of incidence from Western CLL. Splenomegaly is common in African CLL, and this has posed diagnostic problems in differentiating the tumour from malaria-associated hyper-reactive malarial splenomegaly (HMS). One feature of the splenomegalic form of African CLL is that the tumour cells often possess short but fine cytoplasmic projections reminiscent of those observed in Western splenic lymphoma with villous lymphocytes (SLVL). Analysis of Ig VH genes both facilitates discrimination between clonal B-cell tumours and HMS, and reveals the differentiation status of the cell of origin. This study indicated that VH genes of nine cases of clonal splenic B-cell tumours with villous lymphocytes from Ghana were relatively unmutated, consistent with an origin from a naive B cell. These features differ from SLVL which arises from a post-follicular antigen-selected B cell. One possibility is that these splenic B-cell tumours derive from a splenic T-independent B cell, with malaria infection as a potential influence.
Adult↗
Analysis of immunoglobulin E VH transcripts in a bronchial biopsy of an asthmatic patient confirms bias towards VH5, and indicates local clonal expansion, somatic mutation and isotype switch events.
Immunoglobulin E (IgE)-dependent mechanisms play a pivotal role in mediating allergic disease. Previously, VH-Cepsilon transcripts from blood or spleen of atopic asthmatics have been analysed for VH gene usage and patterns of somatic mutation. An over-representation of the minor VH5 family has been observed, consistent with a superantigen drive. As local mucosal events in IgE production may be more significant in the disease process, we have analysed VH-Cepsilon transcripts from a bronchial biopsy of a patient with severe asthma. VH5 predominance was confirmed with 10 of 30 unique clones derived from this family. Repeated sequences, some with intraclonal variation, revealed clonal expansion and continuing mutational activity at the site. Unexpectedly, three unmutated VH-Cepsilon sequences were found, indicating that isotype switching to IgE can occur without mutation. Detection of a sister clone with extensive mutations was again consistent with local mutational activity. Evidence for local isotype switching was obtained by identification of clonally related immunoglobulin M (IgM), immunoglobulin G (IgG) and immunoglobulin E (IgE) sequences. However, in contrast to findings in blood, no IgG4 transcripts clonally related to IgE were detected, suggesting that the balance between synthesis of IgG4 and IgE may differ between systemic and local sites. These data confirm a VH5 bias in IgE, and support the concept that IgE-synthesizing B cells arise via local differentiation.
Adult↗
B cell maturation in relation to multiple myeloma.
Maturation of a normal B-cell precursor to a mature plasma cell involves rearrangement and somatic mutation of the immunoglobulin variable (V) region genes. These events occur at distinct stages of development, and when a B cell undergoes neoplastic transformation, the genetic imprint reflects the clonal history of the cell of origin. Sequence analysis of V-genes can reveal bias as compared with the available repertoire, possibly reflecting a role for superantigen in stimulation of certain B cells. It can also indicate if the tumour cell has encountered the site of somatic mutation in the germinal centre, and if this mechanism is still active post-transformation. Analysis may also reveal a potential influence of persistent antigen in driving tumour growth. Genetic evidence supports the concept that tumour cells are not frozen at a single point of differentiation, but are able to move through certain limited stages. For myeloma, V-gene analysis indicates that the malignant cell population is likely to be derived from an antigen-selected plasma cell, but that a less mature minor B cell population of identical sequence may coexist. In contrast, benign plasma cell tumours can include B cells still undergoing somatic mutation. In both malignant and benign disease, transcripts of clonally-related alternative isotypes have been identified V-gene analysis is contributing to the diagnosis, monitoring and understanding of B-cell tumours, and may facilitate the development of rational approaches to therapy.
B-Lymphocytes↗
A human monoclonal antibody encoded by the V4-34 gene segment recognises melanoma-associated ganglioside via CDR3 and FWR1.
A heterohybridoma cell line producing the human monoclonal antibody (MoAb) MDT.1 has been established. The heavy chain of MoAb MDT.1 is encoded by the VH gene segment V4-34 (previously designated VH4-21), and the light chain is encoded by the V kappa 1-L12a gene segment, both in germline configuration. MDT.1 has reactivity against lipid A, double- and single-stranded DNA, red blood cell associated i antigen, and ganglioside antigens. In a panel of tumour cell lines, MDT.1 reacted specifically with melanoma cells and other tumour cells of neuroectodermal origin. Cellular recognition appears to be via tumour-associated ganglioside antigens, and may involve the minimal essential epitope NeuNac alpha 2-->3Gal beta 1-->-4Glc-. Binding to ganglioside antigen is inhibited by the monoclonal anti-idiotypic antibody 9G4. Since the 9G4 idiotope is located in framework region 1 (FWR1) of V4-34-encoded antibodies, this region is likely to be involved, either directly or indirectly, in ganglioside binding. The complementarity-determining region 3 (CDR3) of MDT.1 is arginine rich, with five out of 12 residues being arginine and these residues are candidates for interaction with the negatively charged ganglioside. The ability of MoAb MDT.1 to recognise ganglioside antigens is associated with potentially useful anti-tumour activity.
Amino Acid Sequence↗
DNA vaccines against haematological malignancies.
DNA vaccines against cancer have to activate an inadequate or damaged immune system in order to attack residual cancer cells. Although the potential problem of tolerance may be overcome by transplantation, provision of high levels of T-cell help is likely to be an important factor in stimulating effective immune pathways. The fusion gene approach appears to provide the required help, and offers a rational design for raising both antibody and T-cell mediated attack against lymphoma and myeloma, which express idiotypic antigen at the cell surface or as a secreted protein respectively. Intriguingly, preliminary data indicate that the fusion gene approach promotes antibody responses against a different cell surface tumour antigen, CEA. Strategies for using DNA vaccines to induce attack on processed peptides bound to MHC class I molecules are also being developed. We hope and anticipate that all categories of tumour antigen may be susceptible to this powerful new technology. The critical clinical requirement, however, will be to treat the presenting tumour with maintenance or restoration of immune capacity. We await results of the preliminary clinical trials with great interest.
Animals↗
DNA vaccination against multiple myeloma.
A variety of approaches to antitumor therapy are currently being explored that use both antigen-encoding DNA and noncoding nucleotides as a component of gene vaccination. Among the specific strategies reviewed are a construct that fuses a single-chain variable fragment (scFv) that incorporates both the variable-region genes necessary to encode the idiotypic determinants with fragment C (FrC) of tetanus toxin; a novel vector system using herpes simplex virus 1 (HSV-1) for in vivo gene delivery; the possibility of eliciting hyperacute xenograft response to treat human cancer; and the use of gene gun-mediated granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA-based tumor cell vaccines. The protection provided by DNA vaccination against viral diseases such as influenza suggested a role for such vaccines against cancer. However, unlike vaccines against infectious diseases, cancer vaccines are therapeutic, rather than prophylactic. With multiple myeloma, for example, it is possible that the optimal timing of administration of such a vaccine is during a remission that has been induced by traditional therapies, to eliminate residual disease. DNA cancer vaccines are designed to activate immune responses to tumor antigens to which the immune system has already been exposed. To do so, the vaccines must first overcome immune tolerance that may have already developed to the tumor. There is increasing evidence that tumor antigens, unlike viral or bacterial antigens, do not consistently activate an immune response. One major factor in determining whether a reaction occurs appears to be whether antigen presentation is accompanied by danger signals. With viral or bacterial infection, the accompanying tissue destruction and inflammation produce costimulatory signals that promote T-cell activation. However, inflammatory and tissue-destructive processes are absent during initial tumor transformation. The typical outcome may be immunologic tolerance.
Genetic Therapy↗
Analysis of VH genes in follicular and diffuse lymphoma shows ongoing somatic mutation and multiple isotype transcripts in early disease with changes during disease progression.
Investigations of VH gene mutational patterns in B-cell tumors are often performed at an arbitrary time point of disease. To assess the effects of disease progression, tumor-derived VH genes have been monitored from presentation through treatment and relapse in one patient with follicle center lymphoma (FCL), and two patients with primary diffuse large B-cell lymphoma (DLCL). The patient with FCL and one patient with DLCL both achieved clinical remission, although this was only partial in the FCL. However, both subsequently relapsed, and the second patient with DLCL was refractory to radiotherapy and chemotherapy. In each case, the tumor-derived VH sequence was identified, and the CDR3 "clonal signature" was used to track tumor cell sequences in subsequent biopsies. All cases showed somatic mutations, with intraclonal heterogeneity evident at presentation, and some sequences were aberrant. The VH sequences of the DLCL which responded to treatment became homogeneous at relapse. The sequences of both the FCL and the refractory DLCL remained heterogeneous. In all cases, transcripts of multiple Ig isotypes could be identified, and there was immunophenotypic evidence for expression of several Ig isotypes. The case of refractory DLCL had identifiable transcripts from IgM, IgD, IgA, IgG, and IgE, but appeared to lose the ability to produce alternative isotype transcripts and protein at the late stage of disease. These cases indicate that VH gene analysis can be used to probe tumor cell behavior in cases of lymphoma and that perturbations caused by therapy and disease progression can occur.
Amino Acid Sequence↗
VH gene analysis of clonally related IgM and IgG from human lymphoplasmacytoid B-cell tumors with chronic lymphocytic leukemia features and high serum monoclonal IgG.
An unusual group of human B-cell tumors with cellular features of chronic lymphocytic leukemia or lymphoplasmacytoid leukemia, together with high levels of a monoclonal IgG serum protein, has been investigated. Analysis of tumor-derived VH genes of neoplastic B lymphocytes was used to determine the clonal relationship between the IgM expressed or secreted by the tumor cells and the IgG serum paraprotein. In all five cases, VH gene sequences showed transcripts of IgM and IgG of common clonal origin. Sequences were derived from VH3 (4 of 5) and VH1 (1 of 5) families and were all highly somatically mutated with strong evidence for antigen selection. There was no intraclonal variation detectable in either IgM or IgG sequences. In 3 of 5 cases, in which monoclonal IgM and IgG were found in serum, the VH genes combined to Cmu or Cgamma showed identical mutational patterns. However, in 2 of 5 cases, in which IgM was confined to cell expression with only monoclonal IgG in serum, sequences of the VH transcripts of IgM and IgG showed many shared mutations but also numerous differences. In these cases, the level of mutation was similar in IgM and IgG and both appeared to be antigen selected. In summary, the final neoplastic event in this group of tumors has apparently occurred at the point of isotype switch from IgM to IgG, leading to dual isotype synthesis. In the group that secreted both isotypes, the mutation pattern was identical, indicating either synthesis by a single cell, or silencing of mutational activity before switching. In the group that did not secrete IgM, cells of each isotype were distinct and reflected a divergent mutational history.
Adult↗
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 C mu, C gamma4 and C epsilon. We have extended this analysis, and have identified V(H)-Cgamma4 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.
Amino Acid Sequence↗