Fernando Díaz-Espada

Anti-idiotypic Immunotherapy in follicular lymphoma patients: results of a long follow-up study.

A specific immune response against the individual idiotypic determinants of clonal immunoglobulins can be elicited in patients with B-cell malignancies. We analyze the clinical outcome and the presence of tumor cells in the blood of 8 patients with follicular lymphoma, vaccinated with autologous idiotype protein from their lymphoma cells. After a median follow-up of 90 months (range: 54 to 128), all patients, except 1, remain in complete clinical remission and 5 are in complete molecular remission. Our results suggest that idiotype vaccination induces long-lasting clinical and molecular remissions and constitutes a potential curative treatment in follicular lymphoma patients.

Intraclonal variability of VH genes in follicular lymphoma patients who have received anti-idiotypic immunotherapy.

Subclonal heterogeneity can affect idiotypic determinants present in the clonotypic immunoglobulin of B-cell follicular lymphomas (FLs) and may limit the effect of antilymphoma treatments performed by immunization of patients with their own tumor-associated idiotypic immunoglobulin. Idiotype-secreting hybridomas were obtained by fusion of tumor cells from 5 patients with FL, and the K6H6/B5 human heteromyeloma and rearranged VH genes from tumor samples and hybridomas were amplified, cloned, and sequenced. Sequences were aligned with germline genes and somatic mutations, intraclonal heterogeneity and genealogic relations of the B-cell clones in the different biopsy specimens were determined. The VH sequence of the progenitor clone was determined in samples of the tumoral population. Further diversification resulted in the presence of 2 to 6 subclones in 4 of the 5 samples studied. Only in 1 patient did the hypermutation mechanism introduce differences among most of the potential idiotopes present in individual subclones. The VH sequence of the hybridoma that provided the idiotypic-vaccine was identified in one of the tumor subclones in all cases. No relapse has been demonstrated in 3 of the 4 vaccinated patients (follow-up: 29-103 months). We conclude that despite potential differences in the idiotypic region expressed by individual tumor cells, at least some potential idiotopes may be preserved among all the tumor subclones in most cases studied. All vaccinated patients developed immune responses against the autologous tumor idiotypic immunoglobulin. Polyclonal anti-idiotypic immune responses induced with a vaccine obtained from 1 hybridoma may be effective against all the idiotypic variants present in the tumor population.

The regulation of membrane-bound and secreted immunoglobulins in the human lymphoid cell line LICR-LON and human hybridomas

The synthesis of membrane-bound and secreted immunoglobulin was investigated in the human line LICR-LON-HMy2, a cell line often used for the derivation of human hybridomas. PAGE-SDS analysis of immunoprecipitates obtained from 35S-methionine labelled cell lysates shows that LICR-LON synthesize a hitherto undetected membrane form of IgG (with a heavy chain of mol. wt 62,000) in addition to the secretor form of IgG already described (55,000 heavy chain). Tunicamycin treatment, pulse-chase experiments and Western blot analysis showed that both chains are synthesized as independent proteins. Hybridomas obtained after fusion of LICR-LON and human peripheral blood lymphocytes retained the ability of the parental cell line to synthesize gamma m and gamma s. Some of these hybrids synthesize and secrete IgM which presumably originates from the parental B-lymphocytes. Precipitation and PAGE-SDS analysis of membrane proteins after iodination of intact cells revealed only one heavy chain band, corresponding in size to that of the gamma m. No indication of the synthesis of the membrane form of IgM was found in the hybrids. These data show that the parental (lymphoid) phenotype (m and s-IgG) is codominant with the more differentiated phenotype (s-IgM) of the fusion partner cell (plasma cell). These observations are compatible with a class-specific m-s regulation operating on a different chromatin structure at the expressed Ig loci of each parental chromosome.

Isolation of tumor-derived immunoglobulin-idiotype from peripheral blood mononuclear cells in a B-cell lymphoma patient with minimal disease.

Summary: Active immunization with autologous idiotypic immunoglobulin, obtained by somatic fusion techniques, has been shown to be a useful alternative treatment in patients with B-cell lymphoma. Nevertheless, the requirement for biopsy specimens to obtain lymphoma cells could be a limitation to this therapeutic strategy. We address the question of whether peripheral blood samples containing small amounts of tumor cells can be used as appropriate fusion partners to rescue tumor-derived idiotypic proteins. In this report, we show that hybrid cells can be obtained from somatic fusions of K6H6/B5 heterohybridoma with lymphoma cells obtained from both lymph node (LN) and peripheral blood mononuclear cells (PBMC) containing only minor amounts of tumor cells. Some hybrid cells obtained from LN or PBMC fusions present an immunoglobulin (Ig) heavy-chain gene rearrangement identical with that of the original tumor and secrete identical Ig protein containing the expected H and L chains.

Effect of purified human interferon-α on the expression of differentiation antigens and mitogen reactivity of cultured human thymic cells

Human thymic cells were cultured in vitro either alone or with the addition of a highly purified preparation of human interferon-alpha. Immunofluorescence techniques using a series of monoclonal antibodies showed that 2-day cultured thymocytes express a more mature phenotype (low HTA 1, high T3 and HLA-A,B,C) than normal, uncultured thymocytes. Interferon addition to the cultures results in a strong increment in the number of HLA+ cells and in the total amount of HLA expressed by the cultured cells. Experiments with purified cell populations showed that the cortical, immature, thymocyte was the target cell for interferon action. Phytohemagglutinin responses--but not interleukin 2 responses--were diminished after pretreatment of thymic cells with interferon. We suggest that interferon may favor a pathway of intrathymic differentiation phenotypically characterized by a high content of Class I HLA antigens.