Immunoglobulin framework-derived peptides function as cytotoxic T-cell epitopes commonly expressed in B-cell malignancies.

Although the idiotypic structures of immunoglobulin from malignant B cells were the first tumor-specific determinants recognized, and clinical vaccination trials have demonstrated induction of tumor-specific immunity, the function of immunoglobulin-specific CD8+ cytotoxic T lymphocytes in tumor rejection remains elusive. Here, we combined bioinformatics and a T cell-expansion system to identify human immunoglobulin-derived peptides capable of inducing cytotoxic T-lymphocyte responses. Immunogenic peptides were derived from framework regions of the variable regions of the immunoglobulin that were shared among patients. Human-leukocyte-antigen-matched and autologous cytotoxic T lymphocytes specific for these peptides killed primary malignant B cells, demonstrating that malignant B cells are capable of processing and presenting such peptides. Targeting shared peptides to induce T-cell responses might further improve current vaccination strategies in B-cell malignancies.

Generation of cytotoxic T lymphocytes against native and altered peptides of human leukocyte antigen-A*0201 restricted epitopes from the human epithelial cell adhesion molecule.

A growing number of human tumor antigens have been described that can be recognized by CTLs in a MHC class I restricted fashion. The epithelial cell adhesion molecule (Ep-CAM) is expressed in a variety of human tumors and has attracted attention as a therapeutic target for monoclonal antibody serotherapy. We have identified immunogenic peptides derived from Ep-CAM, that bind to human leukocyte antigen-A*0201 and elicit strong peptide-specific human CTL responses, demonstrating that there is an effective T-cell repertoire against these Ep-CAM-derived peptides that can be recruited. Alterations to these peptides were made to increase their binding affinity to MHC class I molecules. The use of such "heteroclitic" peptides allowed generation of cytotoxic T cells that demonstrated increased killing of target cells pulsed not only with the heteroclitic but also with the native peptide. Most important, CTL cell lines that are generated against these peptides specifically lyse epithelial tumor cells expressing Ep-CAM but not normal hematopoietic or bronchial epithelial cells.

Detection of clonally rearranged T-cell-receptor gamma chain genes from T-cell malignancies and acute inflammatory rheumatic disease using PCR amplification, PAGE, and automated analysis.

Clonal expansions of T cells carrying identical T-cell-receptor (TCR) genes are the hallmark of T-cell malignancies, but they can also result from a strong immune reaction to a dominant epitope. The basis for the molecular detection of clonal T cells is amplification of the V-(D)-N-J region of the TCR gene. We evaluated PCR amplification of the rearranged gamma TCR from genomic DNA extracted from peripheral blood and subsequent polyacrylamide gel electrophoresis (PAGE) in an automated DNA sequencer. We determined the sensitivity for the detection of clonal T cells and propose a standardized evaluation procedure for the electrophoretic profiles generated by the DNA sequencer. The sensitivity of our method was 0.6-1.25% of clonal T cells within a polyclonal background. Sixteen patients with T-cell malignancies, ten with acute inflammatory rheumatic diseases, and twelve healthy controls were examined. Among the systemic T-cell malignancies, all but one patient with T-PLL (8/ 9) revealed a clonal PCR signal. No clonal signal was detectable in any patient in clinical complete remission (5/5) or in either of the two patients with lymphomas limited to cutaneous sites. However, clonal T cells were detected in one patient with polymyalgia rheumatica and in one with reactive arthritis. A polyclonal signal was found in the remaining eight patients with acute inflammatory rheumatic diseases and in 12 healthy controls. Taking our results together, the PCR/PAGE assay is able to reliably distinguish clonal from polyclonal T-cell populations. However, although the sensitivity is limited to approximately 1%, clonal T cells can be found in the peripheral blood of some patients with autoimmune diseases and not only in T-cell malignancies.

A case of mu heavy-chain disease associated with hyperglobulinemia, anemia, and a positive Coombs test.

We report here for the first time a patient with mu heavy-chain disease (HCD), hyperimmunoglobulinemia, and a positive direct antiglobulin test (DAT, Coombs test). The heavy-chain diseases involve the proliferation of lymphoplasma cells of B cell origin and are characterized by the production of incomplete heavy chains devoid of light chains. The association of mu heavy-chain disease with either hyperglobulinemia or a positive DAT has not been reported in the literature to date. In this patient, immunofixation of serum proteins with monospecific antisera to alpha-, gamma-, mu,- or delta-chains and to kappa- and lambda-chains revealed a precipitation band with antibody to IgM, but not with kappa and lambda light-chain antibodies, indicating mu heavy-chain disease. Hyperglobulinemia was present, which is very uncommon for HCD. A DAT of the patient's red blood cells (RBC) was found to be strongly positive for anti-IgG but negative for anti-IgM, -IgA, -C3c, and -C3d. However, when the eluate from the patient's red blood cells was investigated with nephelometry, it was found to contain antigens reactive with anti-y as well with anti-mu-antiserum. When a DAT was performed with a randomly chosen test cell incubated with the eluate, the antibody-containing eluate was shown to react with anti-IgG as well as with anti-IgM-antiserum. In summary, the eluate from the patient's RBCs contained IgG and an immunoglobulin structure reactive with anti-IgM in an RBC agglutination assay as well as with anti-mu antiserum in a nephelometric investigation. Whether this IgM on the patient's erythrocytes is penta- or oligomeric, complete IgM, or the heavy chain cannot be concluded from these observations.

Successful treatment of bronchiolitis obliterans organizing pneumonia with low-dose methotrexate in a patient with Hodgkin's disease.

Bronchiolitis obliterans organizing pneumonia (BOOP) is a rare disease, which is histopathologically defined by the presence of granulation tissue in the bronchioles, alveolar ducts and alveoli leading to plugging of the bronchiolar and alveolar lumen. BOOP is considered as a nonspecific response to many types of lung injury, including drugs, radiation, an underlying hematologic malignant neoplasm, autoimmune diseases, bacterial or virus infection, or an underlying lung disease, or occurs idiopathically. BOOP is mainly treated with corticosteroids, which induce a rapid clinical improvement. A frequent problem is relapse of disease when corticosteroid dosage is tapered off. We present the case of a 20-year-old patient with Hodgkin's disease developing BOOP after chemotherapy (COPP/ABVD) and irradiation. Initially, she responded well to corticosteroids, but relapsed when medication was discontinued. Complete remission of BOOP was achieved by long-term treatment with low-dose methotrexate (5-20 mg/week, i.v.).

Anti-CD20 antibody as consolidation therapy in a patient with primary plasma cell leukemia after high-dose therapy and autologous stem cell transplantation.

In multiple myeloma (MM), circulating malignant B cells are proposed as the proliferative compartment of the disease. In view of the close relationship between multiple myeloma and primary plasma cell leukemia (PCL), an anti-CD20 antibody treatment might also be considered as consolidation for patients with PCL. A 55-year-old patient diagnosed with PCL achieved complete remission after autologous transplantation. A total of four weekly courses of rituximab (375 mg/m(2)) were administered. Prior to antibody therapy, CD20+ cells comprised 22.6% of the mononuclear cells in peripheral blood (PB) assessed by flow cytometry and were enriched by magnetic activated cell sorting (MACS). In the enriched CD20+ fraction, 0.093% clonotypic cells were detected using a quantitative polymerase chain reaction (PCR) assay based on limiting dilutions. The proportion of clonotypic cells was 0.034% in PB and 0.032% in bone marrow (BM). Rituximab depleted CD20+ cells completely in PB and BM. Tumor load in PB and BM at day 40 and in PB at day 70 did not change in comparison to prior to therapy (0.037% in PB, 0.026% in BM). At day 90, the tumor load increased to 0.066% in PB. At day 120, the patient relapsed with 0.65% CD38++/CD138+/CD20- plasma cells and furthermore no CD20+ B cells in PB. The expansion of plasma cells was accompanied by an increase in the tumor load in both compartments (PB: 0.65%, BM: 1.8%). The accumulation of plasma cells during disease progression without the reappearance of CD20+ cells did not sustain the role of circulating clonotypic B cells as proliferative compartment in our patient. However, it cannot be excluded that rituximab was not able to eradicate malignant B cells, which subsequently contributed to relapse.

Analysis of the T cell response to tumor and viral peptide antigens by an IFNgamma-ELISPOT assay.

We have established a sensitive ELISPOT assay measuring interferon gamma (IFN gamma) release on a single-cell basis to detect influenza peptide-specific CD8+ T cells in uncultured peripheral blood mononuclear cells (PBMC). Using this method, we studied the T cell response to HLA-A1 and HLA-A2.1 binding peptide epitopes derived from the MAGE-1 and MAGE-3 proteins, from the melanoma-associated antigens tyrosinase, Melan-A/MART-1 and gp100, and from influenza proteins in stage IV melanoma patients and healthy controls. In 18 of 24 HLA-A2-positive donors (75%), but only in 9 of 25 HLA-A2-positive melanoma patients (36%) T cells reactive with the influenza matrix peptide were demonstrated (p = 0.007). T cells responding to one or several of the melanoma-associated peptides were detected in 5 of 25 HLA-A2-positive patients with metastatic melanoma. Four of these 5 patients had been treated with interleukin-2- and IFN alpha-containing therapy. Two of the 24 healthy donors had T cells reactive with the MART-1 27-35 peptide. No reactivity with the HLA-A1-binding peptides from MAGE-1 or MAGE-3 was detected in any of the HLA-A1-positive healthy controls or melanoma patients. These results show that the IFN gamma-ELISPOT assay is suitable to determine quantitatively T cells reactive with melanoma-associated and influenza peptide epitopes in uncultured PBMC. The failure to detect T cells responding to influenza in many melanoma patients with progressive disease may indicate an impairment of their T cell function.

Induction of cytotoxic T-cell responses against immunoglobulin V region-derived peptides modified at human leukocyte antigen-A2 binding residues.

Cytotoxic T-lymphocyte (CTL) responses can be generated against peptides derived from the immunoglobulin (Ig) V region in some but not all patients. The main reason for this appears to be the low peptide-binding affinity of Ig-derived peptides to major histocompatibility complex (MHC) class I molecules and their resulting low immunogenicity. This might be improved by conservative amino acid modifications at the MHC-binding residues of the peptides (heteroclitic peptides). In this study, it was found that in 18 Ig-derived peptides, that heteroclitic peptides from the Ig gene with improved binding to human leukocyte antigen (HLA)-A*0201 can be used to improve CTL responses. Amino acid substitution substantially increased predicted binding affinity, and there was a strong correlation between predicted and actual binding to HLA-A*0201. CTLs generated against the heteroclitic peptide had not only enhanced cytotoxicity against the heteroclitic peptide but also increased killing of antigen-presenting cells pulsed with the native peptide. Surprisingly, no difference was observed in the frequency of T cells detected by MHC class I peptide tetramers after stimulation with the heteroclitic peptide compared with the native peptide. CTLs generated against heteroclitic peptides could kill patients' tumor cells, showing that Ig-derived peptides can be presented by the tumor cell and that the failure to mount an immune response (among other reasons) likely results from the low immunogenicity of the native Ig-derived peptide. These results suggest that heteroclitic Ig-derived peptides can enhance immunogenicity, thereby eliciting immune responses, and that they might be useful tools for enhancing immunotherapy approaches to treating B-cell malignant diseases.