Among B cell non-Hodgkin's lymphomas, MALT lymphomas express a unique antibody repertoire with frequent rheumatoid factor reactivity.

We analyzed the structure of antigen receptors of a comprehensive panel of mature B non-Hodgkin's lymphomas (B-NHLs) by comparing, at the amino acid level, their immunoglobulin (Ig)V(H)-CDR3s with CDR3 sequences present in GenBank. Follicular lymphomas, diffuse large B cell lymphomas, Burkitt's lymphomas, and myelomas expressed a CDR3 repertoire comparable to that of normal B cells. Mantle cell lymphomas and B cell chronic lymphocytic leukemias (B-CLLs) expressed clearly restricted albeit different CDR3 repertoires. Lymphomas of mucosa-associated lymphoid tissues (MALTs) were unique as 8 out of 45 (18%) of gastric- and 13 out of 32 (41%) of salivary gland-MALT lymphomas expressed B cell antigen receptors with strong CDR3 homology to rheumatoid factors (RFs). Of note, the RF-CDR3 homology without exception included N-region-encoded residues in the hypermutated IgV(H) genes, indicating that they were stringently selected for reactivity with auto-IgG. By in vitro binding studies with 10 MALT lymphoma-derived antibodies, we showed that seven of these cases, of which four with RF-CDR3 homology, indeed possessed strong RF reactivity. Of one MALT lymphoma, functional proof for selection of subclones with high RF affinity was obtained. Interestingly, RF-CDR3 homology and t(11;18) appeared to be mutually exclusive features and RF-CDR3 homology was not encountered in any of the 19 pulmonary MALT lymphomas studied.

Vector-based vaccine/cytokine combination therapy to enhance induction of immune responses to a self-antigen and antitumor activity.

Many antigens associated with human tumors are overexpressed in tumor cells as compared with normal tissues; these "self" tumor-associated antigens are also expressed during fetal development, and it is, thus, not surprising that they are either weakly immunogenic or functionally nonimmunogenic in the tumor-bearing host. In the studies reported here, we have used different vaccines and vaccine strategies in an attempt to develop antitumor immunity in a stringent animal model. The tumor antigen used was human carcinoembryonic antigen (CEA). The model used was CEA transgenic mice, in which the human CEA transgene is under the control of the endogenous CEA promoter; CEA is expressed in fetal tissues and normal gastrointestinal tissues, and CEA protein is found in sera. Previous studies have shown these CEA transgenic mice to be tolerant to the induction of CEA immunity using CEA protein in adjuvant as an immunogen. CEA-expressing tumor cells were implanted 14 days before vaccine therapy. The vaccines used were recombinant vaccinia virus containing the transgenes for CEA and three T-cell costimulatory molecules [B7-1, ICAM-1, and LFA-3, designated recombinant vaccinia (rV)-CEA/TRICOM], with each transgene under the control of individual poxvirus promoters, and a replication-defective avipox virus (fowlpox; rF) containing the same four transgenes (designated rF-CEA/TRICOM). The results demonstrate that (a) continued boosting with vaccine is required to maintain CEA-specific T-cell responses, and boosting with rF-CEA/TRICOM is superior to boosting with rF-CEA; (b) a diversified vaccination protocol consisting of primary vaccination with rV-CEA/TRICOM followed by boosting with rF-CEA/TRICOM is more efficacious than homogeneous vaccination with rF-CEA/TRICOM in the induction of both CEA-specific T-cell responses and antitumor activity; and (c) the use of cytokines, local granulocyte macrophage colony-stimulating factor (GM-CSF) and low-dose systemic interleukin 2, in combination with vaccine is essential in inducing antitumor activity, as compared with the use of cytokines alone, or the use of vaccines without cytokine. Both GM-CSF and interleukin 2 were shown to contribute to the induction of CEA-specific T-cell responses. These studies thus provide a "proof of concept" that potent vaccines and vaccine strategies, in combination with cytokines, may be essential to obtain the level of T-cell responses directed against a self-antigen that is necessary to achieve antitumor responses.

Modified vaccinia virus ankara recombinants are as potent as vaccinia recombinants in diversified prime and boost vaccine regimens to elicit therapeutic antitumor responses.

Cancer vaccine regimens use various strategies to enhance immune responses to specific tumor-associated antigens (TAAs), including the increasing use of recombinant poxviruses [vaccinia (rV) and fowlpox (rF)] for delivery of the TAA to the immune system. However, the use of replication competent vectors with the potential of adverse reactions have made attenuation a priority for next-generation vaccine strategies. Modified vaccinia Ankara (MVA) is a replication defective form of vaccinia virus. Here, we investigated the use of MVA encoding a tumor antigen gene, carcinoembryonic antigen (CEA), in addition to multiple costimulatory molecules (B7-1, intercellular adhesion molecule-1, and lymphocyte function-associated antigen-3 designated TRICOM). Vaccination of mice with MVA-CEA/TRICOM induced potent CD4+ and CD8+ T-cell responses specific for CEA. MVA-CEA/TRICOM could be administered twice in vaccinia naïve mice and only a single time in vaccinia-immune mice before being inhibited by antivector-immune responses. The use of MVA-CEA/TRICOM in a diversified prime and boost vaccine regimen with rF-CEA/TRICOM, however, induced significantly greater levels of both CD4+ and CD8+ T-cell responses specific for CEA than that seen with rV-CEA/TRICOM prime and rF-CEA/TRICOM boost. In a self-antigen tumor model, the diversified MVA-CEA/TRICOM/rF-CEA/ TRICOM vaccination regimen resulted in a significant therapeutic antitumor response as measured by increased survival, when compared with the diversified prime and boost regimen, rV-CEA/TRICOM/rF-CEA/TRICOM. The studies reported here demonstrate that MVA, when used as a prime in a diversified vaccination, is clearly comparable with the regimen using the recombinant vaccinia in both the induction of cellular immune responses specific for the "self"-TAA transgene and in antitumor activity.

Expression of activation-induced cytidine deaminase is confined to B-cell non-Hodgkin's lymphomas of germinal-center phenotype.

Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination of the immunoglobulin (IG) genes in B cells. It has recently been proposed that AID, as the newly identified DNA mutator in man, may be instrumental in initiation and progression of B-cell non-Hodgkin's lymphomas (B-NHL). We quantitatively measured, by real-time reverse-transcription PCR, expression of AID and of the error-prone DNA polymerase iota in normal B cells and a comprehensive panel of B-NHL entities. In pre- and postgerminal center (GC)-type B-NHLs like in normal naive and memory cells, AID did not exceed background levels. However, half of Burkitt lymphomas tested were found to express AID, at most at levels comparable with those found in normal GC B cells. Thirty percent of diffuse large B-cell lymphomas also transcribed AID, some at supraphysiological levels. Of follicular lymphoma cases, only 25% expressed significant amounts of AID. Moreover, within the group of GC-type B-NHLs, a statistically significant correlation between AID and polymerase iota expression was found. By contrast, we observed no correlation between AID expression and mutation load neither with the degree of intraclonal diversity of IG variable heavy chain genes. Interestingly, in two of seven follicular lymphomas with clinical and histological progression, selective outgrowth of AID-expressing clones occurred, suggestive for a role of the somatic diversification machinery in lymphoma transformation.

Vaccine therapy of established tumors in the absence of autoimmunity.

PURPOSE: Many current clinical trials involve vaccination of patients with vaccines directed against tumor-associated antigens, which are, in actuality, "self-antigens" overexpressed in tumors as compared with normal tissues. As tumor vaccines become more potent through the addition of costimulatory molecules and cytokines and the use of diversified prime and boost regimes, the level of concern rises regarding the balance between antitumor immunity and pathological autoimmunity. Studies were conducted using mice bearing a transgenic self-antigen [human carcinoembryonic antigen (CEA)], which is expressed in some normal adult tissues, and tumor expressing the same self-antigen. These mice were vaccinated with recombinant poxviral vectors [recombinant vaccinia, recombinant fowlpox (rF)] encoding the CEA transgene as well as a triad of costimulatory molecules [B7-1, ICAM-1, and LFA-3 (TRICOM)]. Here we investigate the mechanism of tumor therapy and evaluate the safety of such a regimen in a self-antigen system. To our knowledge, the study reported here is the first description of a vaccine to a defined antigen where the regimen is potent enough to induce tumor therapy in the absence of autoimmunity. EXPERIMENTAL DESIGN: CEA transgenic mice were transplanted with CEA-expressing tumors. Fourteen days later, mice were vaccinated with recombinant vaccinia-CEA/TRICOM admixed with recombinant murine granulocyte macrophage colony-stimulating factor and then given low-dose interleukin 2. Mice were boosted on days 21, 28, and 35 with rF-CEA/TRICOM admixed with rF-granulocyte macrophage colony-stimulating factor and then given low-dose interleukin 2. Mice were monitored for survival and compared with groups of mice vaccinated in a similar manner with poxviral vectors containing CEA/B7-1 or CEA transgenes. To determine the mechanism of antitumor therapy, mice were depleted of T-cell subpopulations before vaccination with the CEA/TRICOM regimen. Mice successfully cured of tumor and age-matched control mice were monitored for 1 year. At 1 year, several clinical assays were carried out involving analysis of 9 serological parameters, 11 urinalysis parameters, and 14 immunological parameters. In addition, histopathology was performed on 42 tissues/mouse. RESULTS: The CEA/TRICOM vaccination regimen induced a therapeutic antitumor response as measured by increased survival, which was due largely to induced T-cell responses (both CD4(+) and CD8(+)) as determined by selective T-cell subset depletion. The CEA/TRICOM vaccination regimen induced a significant increase in proliferation of CD4(+) T cells to CEA protein and a significant increase in secretion of IFN-gamma from CD8(+) T cells in response to a defined CEA epitope. Despite CEA expression in normal adult gastrointestinal tissues, no toxicity was observed in the CEA/TRICOM-vaccinated group when an array of clinical serum and urine chemistry assays was conducted 1 year after vaccination. Moreover, a comprehensive histopathological evaluation of all tissues from these groups also showed no evidence of toxicity. CONCLUSIONS: Activation of T cells directed against a tumor-associated self-antigen, sufficient to mediate therapeutic antitumor immunity, was observed in vivo without the development of autoimmunity as analyzed by a comprehensive evaluation of biochemical, immunological, and histopathological criteria. These studies demonstrate that the use of vectors containing as many as three costimulatory molecules does not induce autoimmunity or other pathology. These studies thus demonstrate that a balance can indeed be achieved between the induction of an immune response to a self-antigen, which is capable of antitumor therapy, and the absence of autoimmunity.

In situ analysis of the variable heavy chain gene of an IgM/IgG-expressing follicular lymphoma: evidence for interfollicular trafficking of tumor cells.

It is generally assumed that follicular lymphomas (FL) not only morphologically resemble normal germinal centers but have retained some functional characteristics of their non-neoplastic counterparts as well. Recent IgV gene analyses on a panel of FLs however, strongly suggested that FLs do not retain the capacity of somatic hypermutation and are not being selected on basis of the quality of their mIgV regions. To extend these findings, we investigated the follicular organization and class switching in a FL that consisted of both IgM- and IgG-expressing tumor cells with a high somatic mutation load and significant intraclonal V(H) gene diversity. V(H)-C(mu) and V(H)-Cgamma gene transcripts were amplified and sequenced from samples of approximately 50 tumor cells, isolated from frozen tissue sections by laser microdissection. We identified many different subclones and obtained limited evidence of subclone dominance in individual follicles. Remarkably, several subclones were found scattered over different follicles. All samples contained IgM- and IgG-expressing tumor cells with, in general, non-identical mutation patterns, which is not in support of ongoing class switching. Accordingly, no switch circle recombination products were found. The findings indicate that the neoplastic follicles lack the organization and functions typical of reactive germinal centers.

Follicular lymphoma with a novel t(14;18) breakpoint involving the immunoglobulin heavy chain switch mu region indicates an origin from germinal center B cells.

With the use of DNA-fiber fluorescent in situ hybridization, a BCL2 protein positive follicular lymphoma with a novel BCL2 breakpoint involving the immunoglobulin heavy chain (IGH) switch mu (S(mu)) region instead of the J(H) or D(H) gene segments was identified. Sequence analysis showed that the genomic breakpoint is localized between the S(mu) region of the IGH complex and the first intron of BCL2. Reverse-transcriptase polymerase chain reaction showed expression of a unique hybrid IGH-BCL2 transcript involving the transcription initiation site I(mu). Sequence analysis of the V(H) region of the functional nontranslocated IGH allele showed multiple shared somatic mutations but also a high intraclonal variation (53 differences in 15 clones), compatible with the lymphoma cells staying in or re-entering the germinal center. This is the first example of a t(14;18) translocation that results from an illegitimate IGH class-switch recombination during the germinal center B-cell stage.

Primary follicular lymphoma of the small intestine: alpha4beta7 expression and immunoglobulin configuration suggest an origin from local antigen-experienced B cells.

Primary follicular lymphoma of the gastrointestinal tract (GI-FL) is a rare so far poorly studied entity. We analyzed four FL cases located in the small intestine and duodenum to gain insight in their pathogenesis and to find an explanation for their low tendency to disseminate outside the GI tract. GI-FLs resemble nodal FLs with respect to morphology and expression of typical GC markers such as CD10, CD38, and BCL-6. We established that the high levels of the anti-apoptosis protein BCL-2 in the tumor cells are in all cases due to a t(14;18) involving the immunoglobulin heavy chain and BCL-2 loci. Detailed immunoglobulin gene analyses on microdissected tissue samples further supported the GC-cell derivation: GI-FLs carry extensively mutated variable heavy-chain genes. The mutation patterns indicated that at some time point in development stringent antigen receptor-based selection processes must have occurred. Interestingly, three of four neoplasms expressed surface IgA, an immunoglobulin class typical of the mucosal immune system and seldom found in nodal FL. In contrast to nodal FLs, the GI-FLs expressed the alpha4beta7 integrin, an established mucosa-homing receptor also expressed by normal intestinal B and T lymphocytes and by low-grade mucosa-associated lymphoid tissue lymphomas. However, the chemokine receptor CXCR3, expressed on low-grade mucosa-associated lymphoid tissue lymphomas, was not detected on the GI-FLs or on nodal FLs. The combined data suggests that primary FL of the small intestine is a distinct entity that originates from local antigen-responsive B cells.