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Biomedical subjects

Freda K Stevenson

Publications and source records attributed to Freda K Stevenson.

At least 19 recordsLinked to original sources
PubMed2006

Human follicular lymphoma cells contain oligomannose glycans in the antigen-binding site of the B-cell receptor.

Expression of surface immunoglobulin appears critical for the growth and survival of B-cell lymphomas. In follicular lymphoma, we found previously that the Ig variable (V) regions in the B-cell receptor express a strikingly high incidence of N-glycosylation sequons, NX(S/T). These potential glycosylation sites are introduced by somatic mutation and are lymphoma-specific, pointing to their involvement in tumor pathogenesis. Analysis of the V region sugars from lymphoma-derived IgG/IgM reveals that they are mostly oligomannose and, remarkably, are located in the antigen-binding site, possibly precluding conventional antigen binding. The Fc region contains complex glycans, confirming that the normal glycan processing pathway is intact. Binding studies indicate that the oligomannose glycans occupying the V regions are accessible to mannose-binding lectin. These findings suggest a potential contribution to lymphoma pathogenesis involving antigen-independent interaction of surface immunoglobulin of the B-cell receptor with mannose-binding molecules of innate immunity in the germinal center.

Binding Sites↗
PubMed2006

Prime-boost with alternating DNA vaccines designed to engage different antigen presentation pathways generates high frequencies of peptide-specific CD8+ T cells.

The route for presentation of Ag to CD8+ or CD4+ T cells following DNA vaccination is critical for determining outcome, but the pathways involved are unclear. In this study, we compare two different DNA vaccine designs aimed to elicit CD8+ T cell responses against a specific peptide-epitope either by direct- or cross-presentation. Each carries sequences from tetanus toxin (TT) to provide essential CD4+ T cell help. In the first already proven design, the peptide-epitope is fused to the N-terminal domain of fragment C from TT. This appears to act mainly by cross-presentation. In the second design, the peptide-epitope is encoded by a minigene, with induction of Th responses mediated by coexpression of a hybrid invariant chain molecule, incorporating a single determinant from TT (p30) in exchange for class II-associated invariant chain peptide. This design appears to act mainly via direct presentation from transfected APCs. Both vaccines mediated Th-dependent priming of CD8+ T cells in mice, but the kinetics and level of the responses differed markedly, consistent with engagement of distinct pathways of Ag presentation. Importantly, the vaccines could be combined in an alternating prime-boost regime, in either order, generating substantially expanded memory CD8+ T cells, with potent effector function. Taken together, these results demonstrate that vaccination protocols involving different modes of Ag presentation at prime and boost can significantly improve the effectiveness of immunization.

Animals↗
PubMed2006

DNA fusion vaccines induce epitope-specific cytotoxic CD8(+) T cells against human leukemia-associated minor histocompatibility antigens.

The graft-versus-leukemia effect of allogeneic stem-cell transplantation is believed to be mediated by T-cell recognition of minor histocompatibility antigens on recipient cells. For minor histocompatibility antigens HA-1 and HA-2, normal cell expression is restricted to hemopoietic cells, and boosting the immune response to these antigens may potentiate graft-versus-leukemia effect without accompanying graft-versus-host disease. To increase efficacy, expansion of HA-1- or HA-2-specific CTL before transplantation is desirable. However, primary HA-1- or HA-2-specific CTL expanded in vitro are often of low avidity. An alternative approach is to prime specific CTL responses in vivo by vaccination. Clearly, donor vaccination must be safe and specific. We have developed DNA fusion vaccines able to induce high levels of epitope-specific CTL using linked CD4(+) T-cell help. The vaccines incorporate a domain of tetanus toxin (DOM) fused to a sequence encoding a candidate MHC class I binding peptide. This design generates antitumor CD8(+) T-cell responses and protective immunity in preclinical models. For clinical application, we constructed vaccines encoding HLA-A*0201-restricted peptides from human HA-1 and HA-2, which were fused to DOM, and tested their performance in HLA-A*0201-transgenic mice. Priming induced epitope-specific, IFNgamma-producing CD8(+) T cells with cytotoxic function boosted to high levels with electroporation. Strikingly, these mouse T cells efficiently killed human lymphoblastoid cell lines expressing endogenous HA-1 or HA-2. High avidity is indicated by the independence of cytolysis from CD8/MHC class I interaction. These safe epitope-specific vaccines offer a potential strategy to prime HA-1- or HA-2-specific CTL in transplant donors before adoptive transfer.

Amino Acid Sequence↗
PubMed2006

Immunoglobulin heavy chain locus events and expression of activation-induced cytidine deaminase in epithelial breast cancer cell lines.

When cells transform, phenotypic and genetic profiles can be dramatically altered. Nevertheless, a recent report identifying IgG in breast cancer cells was unexpected, revealing differentiation features normally associated with B lymphocytes. To extend these findings, we focused on immunoglobulin variable (V) region gene analysis using well-defined breast cancer cell lines expressing the epithelial marker, epithelial cell adhesion molecule (EpCAM). V(H) gene transcripts were identifiable by nested reverse transcription-PCR either as single or dual V, diversity (D), and joining (J) rearrangements in four of six lines, most being potentially functional. V(D)J transcripts were observed in sequential cultures, indicating stable expression. To exclude coexisting lymphocytes, each cell line was shown to be EBV negative, with CD19/CD20 and cytoplasmic/surface immunoglobulin also absent by flow cytometry. Identified V(H) transcripts were then sought in individual tumor cells, isolated as EpCAM+ single cells by flow cytometry. Importantly, in three of three selected cell lines, V(H) genes were identifiable in a significant fraction (approximately 32%) of single cells. In five of six identified V(H) genes, somatic mutations were apparent with no intraclonal variation, indicating cessation of mutational activity. V(H) transcripts were pre- and post-isotype switch, with activation of switch events evident from expressed germ-line switch transcripts in two of six lines. Strikingly, six of six cell lines expressed activation-induced cytidine deaminase (AID) essential for mutational and switch activity. These data suggest either a de novo rearrangement and modification of V(H) genes in epithelial tumor cells or assimilation of lymphocyte-derived chromatin. Constitutive AID activation in malignant epithelial cells further raises a potential for inducing aberrant mutational activity.

Amino Acid Sequence↗
PubMed2006

Structural and functional features of the B-cell receptor in IgG-positive chronic lymphocytic leukemia.

PURPOSE: To determine the origin and relationship of the rare IgG+ variant of chronic lymphocytic leukemia (CLL) to the two common IgM+IgD+ subsets that are distinguished by expression of unmutated or mutated V(H) genes, with the former having a worse prognosis. EXPERIMENTAL DESIGN: IgG+ CLL cells were characterized using phenotypic, functional, and immunogenetic analyses. RESULTS: IgG+ CLL was phenotypically similar to mutated IgM+IgD+ CLL (M-CLL) and variably expressed CD38 (4 of 14). ZAP-70, a tyrosine kinase preferentially expressed in unmutated CLL, was found in only 2 of 14 cases. The ability to signal via surface IgM (sIgM) varies between the main subsets of CLL and is associated with expression of ZAP-70. In IgG(+) CLL, 9 of 14 responded to engagement of sIgG with no apparent requirement for expression of CD38 or ZAP-70. However, signal capacity correlated with intensity of sIgG expression. Most switched immunoglobulin variable region genes were somatically mutated without intraclonal variation, and no case expressed activation-induced cytidine deaminase. Derivation from a postgerminal center B cell is, therefore, likely, and a relationship with M-CLL is suggested. This is supported by a shared biased usage of the V4-34 gene. Similar bias in normal B cells developed with age, providing an expanded population for transforming events. However, conserved sequences detected in the CDR3 of V4-34-encoded gamma chains were not found M-CLL, indicating no direct path of isotype switch from M-CLL. CONCLUSION: IgG+ CLL is likely to arise from an age-related expanded pool of B cells, on a path parallel to M-CLL, and perhaps with a similar clinical course.

ADP-ribosyl Cyclase 1↗
PubMed2006

Optimizing cancer immunotherapy trials: back to basics.

Attempts to raise effective immunity against cancer are benefiting from information on the nature of the immunity involved and its regulation and, perhaps, now it is time to step back and define our approach in molecular terms prior to clinical testing. Although there are immunological differences between mice and patients, results from murine studies are encouraging early 'translation' of concepts to the clinic and it is vital to take immunological principles emerging from mice into clinical vaccine design. One is the requirement to break tolerance against over-expressed self-antigens, a potentially risky procedure but necessary for several cancer targets. A study in this issue of the European Journal of Immunology attempts to do this by using xenogeneic antigens, albeit with variable outcome. The unstated goal is to activate T-cell help but this can be achieved more effectively by harnessing a predictable anti-microbial repertoire. The second issue lies in the delivery of antigen. One strategy is "prime/boost" using DNA priming and boosting with a viral vector; however, this induces blocking immunity against viral proteins, and must be used judiciously. There are other physical methods to increase immunity such as electroporation, which can itself be used in 'prime/boost' sequence. These twin problems of engagement of T-cell help and delivery of adequate antigen can now be addressed by applying immunological logic to cancer vaccines.

Antigens, Neoplasm↗
PubMed2005

Vaccination of human subjects expands both specific and bystander memory T cells but antibody production remains vaccine specific.

Human subjects maintain long-term immunologic memory against infective organisms but the mechanism is unclear. CD4+ T-helper memory (Thmem) cells are pivotal in controlling humoral and cellular responses, therefore their longevity and response to vaccination are critical for maintenance of protective immunity. To probe the dynamics of the Thmem-cell response to antigenic challenge, we investigated subjects following a booster injection with tetanus toxoid (TT). Expansion of TT-specific Thmem cells and cytokine production showed complex kinetics. Strikingly, parallel expansion and cytokine production occurred in pre-existing Thmem cells specific for 2 other common antigens: purified protein derivative of tuberculin and Candida albicans. Bystander expansion occurred in Thmem but not in Thnaive cells. Antibody production against TT peaked approximately 2 weeks after vaccination and gradually declined. However, pre-existing antibody against the other antigens did not change. It appears that although all Thmem cells are readily stimulated to expand, antibody responses are controlled by antigen availability. These findings relate to the maintenance of memory and have consequences for assessments of specific T-cell responses to vaccination.

Adult↗
PubMed2005

Inhibition of a vaccine-induced anti-tumor B cell response by soluble protein antigen in the absence of continuing T cell help.

DNA vaccination can elicit the production of anti-tumor antibodies, thus obviating the need to continuously administer passive antibody. This vaccination strategy is particularly important where antibodies have proven to be effective anti-tumor agents. To amplify antibody responses against weak tumor antigens, we previously designed DNA-fusion vaccines incorporating tumor sequences linked to microbial genes. By using a safe idiotypic (Id) antigen from a B cell tumor fused to a fragment C (FrC) sequence from tetanus toxin, we induced both anti-Id and anti-FrC antibodies. It was important to determine whether the antigen itself, either injected or released from residual tumor cells, would boost the antibody response. Id protein not only failed to boost the response, but permanently and rapidly inhibited it by ablating Id-specific memory B cells. In contrast, an Id protein-FrC conjugate boosted both Id-specific and FrC-specific responses. Strikingly, the depletion of CD4+ T cells converted the Id protein-FrC conjugate vaccine into an inhibitor. These findings support the hypothesis that the activation of memory B cells by a DNA vaccine encoding a protein antigen, in the presence of the protein itself, depends completely on T cell help. Furthermore, by using knockout mice, we have shown that inhibition of the Id-specific memory B cells by the Id protein is largely independent of the FcgammaRIIB and, hence, independent of immune complexes. The principles revealed by using a DNA vaccine have implications for all cancer vaccines designed to induce and maintain antibody responses against weak autologous tumor antigens.

Animals↗
PubMed2005

Electroporation as a "prime/boost" strategy for naked DNA vaccination against a tumor antigen.

We have developed novel DNA fusion vaccines encoding tumor Ags fused to pathogen-derived sequences. This strategy activates linked T cell help and, using fragment C of tetanus toxin, amplification of anti-tumor Ab, CD4(+), and CD8(+) T cell responses is achievable in mice. However, there is concern that simple DNA vaccine injection may produce inadequate responses in larger humans. To overcome this, we tested electroporation as a method to increase the transfection efficiency and immune responses by these tumor vaccines in vivo in mice. Using a DNA vaccine expressing the CTL epitope AH1 from colon carcinoma CT26, we confirmed that effective priming and tumor protection in mice are highly dependent on vaccine dose and volume. However, suboptimal vaccination was rendered effective by electroporation, priming higher levels of AH1-specific CD8(+) T cells able to protect mice from tumor growth. Electroporation during priming with our optimal vaccination protocol did not improve CD8(+) T cell responses. In contrast, electroporation during boosting strikingly improved vaccine performance. The prime/boost strategy was also effective if electroporation was used at both priming and boosting. For Ab induction, DNA vaccination is generally less effective than protein. However, prime/boost with naked DNA followed by electroporation dramatically increased Ab levels. Thus, the priming qualities of DNA fusion vaccines, integrated with the improved Ag expression offered by electroporation, can be combined in a novel homologous prime/boost approach, to generate superior antitumor immune responses. Therefore, boosting may not require viral vectors, but simply a physical change in delivery, facilitating application to the cancer clinic.

Adjuvants, Immunologic↗
PubMed2005

Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax.

BACKGROUND: Previously, antigens expressed from DNA vaccines have been fused to the VP22 protein from Herpes Simplex Virus type I in order to improve efficacy. However, the immune enhancing mechanism of VP22 is poorly understood and initial suggestions that VP22 can mediate intercellular spread have been questioned. Despite this, fusion of VP22 to antigens expressed from DNA vaccines has improved immune responses, particularly to non-secreted antigens. METHODS: In this study, we fused the gene for the VP22 protein to the gene for Protective Antigen (PA) from Bacillus anthracis, the causative agent of anthrax. Protective immunity against infection with B. anthracis is almost entirely based on a response to PA and we have generated two constructs, where VP22 is fused to either the N- or the C-terminus of the 63 kDa protease-cleaved fragment of PA (PA63). RESULTS: Following gene gun immunisation of A/J mice with these constructs, we observed no improvement in the anti-PA antibody response generated. Following an intraperitoneal challenge with 70 50% lethal doses of B. anthracis strain STI spores, no difference in protection was evident in groups immunised with the DNA vaccine expressing PA63 and the DNA vaccines expressing fusion proteins of PA63 with VP22. CONCLUSION: VP22 fusion does not improve the protection of A/J mice against live spore challenge following immunisation of DNA vaccines expressing PA63.

Journal Article↗
PubMed2005

VP22 enhances antibody responses from DNA vaccines but not by intercellular spread.

In some species DNA vaccines elicit potent humoral and cellular immune responses. However, their performance in humans and non-human primates is less impressive. There are suggestions in the literature that an increase in the intercellular distribution of protein expressed from a DNA vaccine may enhance immunogenicity. We incorporated the Herpes Simplex Virus type 1 (HSV) VP22 gene, which encodes a protein that has been described as promoting intercellular spread, into a DNA vector in which it was fused to enhanced green fluorescent protein (EGFP). Following transfection of the plasmid DNA into mammalian cells, distribution of the fusion protein VP22-EGFP was not increased compared to EGFP alone. Furthermore, we found no evidence to suggest that VP22 was capable of mediating intercellular spread. However, when these constructs were used as DNA vaccines to immunise mice, antibody levels specific to EGFP were significantly enhanced when EGFP was fused to VP22. These data suggest that amplification of the immune response may occur via mechanisms other than VP22-mediated intercellular spread of antigen.

Adjuvants, Immunologic↗
PubMed2005

Update on cancer vaccines.

PURPOSE OF REVIEW: Vaccination against cancer has had a variable history, with claims of success often fading into disappointment. The reasons for this include poor vaccine design, inadequate understanding of the nature of the immune response, and a lack of objective measures to evaluate performance. The impact of genetic technology has changed everything. We now have multiple strategies to identify candidate tumor antigens, and we understand more about activation and regulation of immunity against cancer. There are novel vaccine strategies to activate specific attack on tumor cells. We also have modern assays using surrogate markers of performance to correlate with clinical effects. It is timely to select significant relevant papers to illustrate the growing potential for patients with cancer. RECENT FINDINGS: Recent findings include tumor antigen discovery and vaccine formulation, relevant knowledge concerning mechanisms of induction of effective immunity from preclinical models, and translation into clinical trials with objective evaluation of performance. SUMMARY: The ability of the immune response to dispose of cancer cells is clear. Passive transfer of antibody or immune cells is already clinically successful. We are now in a position to harness new gene-based information to design vaccines capable of inducing effective and long-lasting immunity. Safe vaccines could be used either in patients or in transplant donors. Pilot clinical trials are the means of testing performance, with continuing vaccine design modification to target specific antigens in different cancers.

Animals↗
PubMed2005

Bodyguards and assassins: Bcl-2 family proteins and apoptosis control in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is the most common B-cell malignancy in the Western world and exists as subtypes with very different clinical courses. CLL is generally described as a disease of failed apoptosis. Apoptosis resistance may stem from a combination of microenvironmental survival signals as well as from intrinsic alterations in the apoptotic machinery within the CLL cell. The molecular mechanism involved in controlling apoptosis in CLL is complex and is influenced by many factors, including Bcl-2 family proteins, which are critical regulators of cell death. Here we review the significance of apoptosis dysregulation in CLL, focusing on the role of Bcl-2 and related Bcl-2 family proteins, such as Bax and Mcl-1. The differential properties of the newly described subsets of CLL are also highlighted.

Apoptosis↗
PubMed2005

Identification and assembly of V genes as idiotype-specific DNA fusion vaccines in multiple myeloma.

Tumor-specific markers are important in identifying and tracking malignant cells. In this regard, functionally rearranged immunoglobulin variable (V) region genes in B-cell tumors fulfill and extend these criteria. V genes provide signature motifs in tumor cells and can delineate critical features of the clonal history of the cell of origin. They also define a tumor-specific antigen, which can be targeted for immunotherapy. Our focus has been on using novel DNA fusion vaccines to induce antitumor immunity. Here, we describe in detail the methods for identifying tumor-derived V genes at the nucleotide level in the malignant plasma cells of multiple myeloma. We further present the methodology for assembly of tumor V genes as single-chain variable region fragments (scFv), fused in frame with an immunopotentiating nontoxic bacterial sequence, Fragment C (FrC) of tetanus toxin. These scFv.FrC DNA vaccines provide protection in myeloma models and are currently in clinical trials. The vaccines are patient specific and can be rapidly assembled for clinical use.

Base Sequence↗
PubMed2005

Determining mutational status of immunoglobulin v genes in chronic lymphocytic leukemia: a useful prognostic indicator.

Determining the clonal origins of malignant B-cells will have an impact on disease understanding and management. In this regard, immunoglobulin variable (V) region gene analysis already is having a significant impact in delineating the tumor cell of origin. It can identify, among other features whether such a cell has undergone somatic mutation, which usually occurs within germinal centres. Remarkably, in chronic lymphocytic leukemia (CLL), the mutational status of V genes has allowed researchers to identify two subsets of disease, one originating from an unmutated B-cell with a markedly poorer disease outcome and the other from a mutated B-cell, which associates with long-term survival. The V gene status in CLL thus provides a robust indicator of disease outcome, which is beginning to shape clinical treatment. This chapter describes in detail the methodology for determining V gene usage in CLL, from acquisition of patient sample to generating the V-gene readout.

Amino Acid Sequence↗
PubMed2005

Idiotype gene rescue in follicular lymphoma.

Beyond the morphological, immunophenotypic, and genetic information used for the diagnosis of lymphoid malignancies, molecular analyses have deepened our insights into the development of B-cell lymphomas. We have learned that B-cell tumors can be grouped according to the mutational status of their immunoglobulin variable (V) region genes, and this has become an important prognostic tool in chronic lymphocytic leukemia. The analysis of V genes also has allowed us to more precisely place B-cell lymphomas relative to their normal B-cell counterparts and to the germinal center where somatic hypermutation takes place. It has become evident that many of the common B-cell tumors arise at this site and are able to respond to stimuli, which govern normal B-cells. In this chapter, we focus on the analysis of V genes in follicular lymphomas based on the experience in our laboratory and provide a detailed guide for this analysis.

B-Lymphocytes↗