Lymphocyte subset alterations in nodes regional to human melanoma.

The lymphocyte subpopulations in tumor-draining lymph nodes of melanoma patients were determined using two-color flow cytometry. Data were analyzed according to: (a) the staging of the melanoma; (b) whether or not the nodes contained tumor; and (c) their distance from the primary tumor. Compared with Stage I patients (without metastasis), uninvolved nodes of stage II patients (with nodal metastases) had a significant decrease in helper/inducer (CD4+) T-cells (P less than 0.001), with a corresponding increase in cytotoxic/suppressor (CD8+) cells (P less than 0.001) and Leu 19+ natural killer (CD56+) cells (P less than 0.01). In some patients the presence of tumor within a node was associated with a large decrease in CD3+ total T-cells, whereas in others tumor involvement had little influence on lymphocyte phenotypes. When analyzed by distance from the primary tumor, nodes closest to tumor in Stage I patients contained a smaller percentage of CD19+ B-cells. In Stage II, tumor-free nodes nearest to tumor showed an increase in CD19+ cells, but statistical significance was not reached. CD56+ natural killer cells increased progressively in nodes near tumor and were more numerous in Stage II uninvolved nodes compared with Stage I nodes. Alterations in phenotypically defined lymph node lymphocytes occur in nodes regional to melanoma as the disease progresses, as growth of metastases occurs, and in tumor-free nodes nearest to tumor. These alterations may be essential to the establishment and progression of metastases.

Measurement of human and mouse anti-tetanus antibodies and isotype analysis by ELISA.

A rapid and sensitive enzyme immunoassay (ELISA) was developed for the quantitation of anti-tetanus antibodies. This technique was used to measure antibody levels in the plasma of immunized donors, in human anti-tetanus IgG preparations and in human and mouse hybridomas producing monoclonal antibodies to tetanus toxoid. The assay was capable of detecting antibody levels as low as 5 X 10(-4) IU/ml. By inclusion of an extra step involving antibodies to mouse Ig isotypes, a sandwich enzyme immunoassay (SEI) was developed which permitted determination of the Ig isotype of mouse anti-tetanus antibodies including tetanus-specific mouse monoclonal antibodies. SEI confirmed Protein A-Sepharose fractionation of mouse ascites fluid containing anti-tetanus antibody. The tetanus toxoid-coated plates have a shelf life of at least 1 year.

Isotype responses of mice to tetanus-toxoid preparations.

The development of anti-tetanus antibodies in CBA/Ca and Balb/c mice immunised with two tetanus-toxoid preparations has been investigated by sensitive enzyme-linked immunoassays. These studies revealed the presence in both strains of mice of naturally occurring antibodies to tetanus toxoid. These were of the IgM, IgA and, to a lesser extent, the IgG3 isotypes. Differences in the responses of mice to the two toxoid preparations were noted; the purer preparation elicited a more rapid and pronounced response. Both strains of mice exhibited similar isotype responses.

Lymphocytes from lymph nodes at different distances from human melanoma vary in their capacity to inhibit/enhance tumor cell growth in vitro.

Eighty-six lymph nodes at measured distances from a primary tumor were removed from 68 patients with malignant melanoma. The ability of lymph-node lymphocytes (LNL) from these nodes to modulate proliferation of a melanoma cell line (UCLA-SO-M14) in vitro was tested. LNL from the majority of lymph nodes (66%) inhibited M14 growth, but LNL from 34% of nodes stimulated growth of the cell line. Peripheral blood mononuclear cells always inhibited M14 growth. Distance of a node from the primary tumor was found to be an important determinant of LNL activity. This was demonstrated using pairs of nodes from individual patients. In 86% of cases, LNL from nodes located nearer to the tumor inhibited M14 growth less than LNL from more distant nodes. Stimulation of M14 growth was commonest with LNL from nodes located near to the tumor. CD8+ T cells were largely responsible for M14 growth inhibition, whereas CD4+ cells were associated with stimulation of M14 growth. Removal of CD4+ lymphocytes from growth stimulatory LNL resulted in a CD4-depleted LNL preparation that inhibited M14 cell proliferation. The environment in lymph nodes located dose to tumors may thus favor growth of metastatic tumor cells.

Immunosuppression by melanoma cells as a factor in the generation of metastatic disease.

Studies of the regional nodes (RLN) of melanoma patients, using immunohistology with anti-S-100 protein and monoclonal antibodies have shown occult tumor cells (OTC) in nodes ostensibly tumor-free by H&E staining. OTC were demonstrated in 14% of Stage I patients, mainly those with deep, thick primaries and 30% of Stage II patients, mainly those with at least 3 tumor-positive nodes on H&E. The nodes containing OTC are those nearest to tumor on the direct lymphatic pathway (dye studies). Parallel studies show nodes in the same position to be immune suppressed (histology, immunohistology, response to mitogens, alloantigens and lymphokines) and to contain many suppressor T cells (Con-A). Melanoma-derived materials (gangliosides, prostaglandins, lipoprotein antigens) downregulate lymphocyte and macrophage functions, providing a possible mechanism for the suppressed function of nodes near tumor, a suppression that may facilitate tumor cells as evidenced by the survival of OTC.

Experiences in the production of human monoclonal antibodies to tetanus toxoid.

The production of human monoclonal antibodies has been attempted using tetanus toxoid as a model antigen since a human antibody of this specificity could have clinical applications. Both mouse and human myeloma cell lines were used as fusion partners and the effects of in vivo and in vitro antigen boosting were also investigated. A cell line, ES12, which secreted specific tetanus toxoid antibody arose following fusion of lymphocytes from a donor who had been boosted three months earlier. Antibody was secreted by this cell line at a high titre (2.5 I.U/ml); it was of IgG isotype and it protected mice against challenge with tetanus toxin. This is, therefore, a first step in producing a therapeutically useful monoclonal antibody.