The staphylococcal toxins are responsible for a number of diseases in man and other animals. Many of them have also long been known to be powerful T cell stimulants. They do not, however, stimulate all T cells. On the contrary, each toxin reacts with human T cells bearing particular V beta sequences as part of their receptors for major histocompatibility complex protein-associated antigen. The specificity of these toxins for V beta s puts them in the recently described class of superantigens and may account for the differential sensitivity of different individuals to the toxic effects of these proteins.
Progress in defining the nature of T cell receptors for antigen has been slow and many conflicting results have been reported (reviewed in reference 1). Recently more success has been achieved by developing monoclonal antibodies, directed against T cell clones, that show unique specificities for the clones (2-4). In this study we have utilized a similar approach and have screened monoclonal antibodies made against cells from a human T cell leukemia for idiotypelike specificity. Two such monoclonal antibodies were found that reacted with the same membrane molecule; one showed absolute specificity for the immunizing cells, the other also reacted with a small population of normal T cells. Materials and MethodsLeukemic cells were obtained from a male from the Dominican Republic. He presented with typical clinical and laboratory findings of Sezary Syndrome. Peripheral leukocyte counts ranged from 30,000 to 97,000 with -~85% Sezary cells. The patient was leukopheresed and leukemic cells obtained by Ficoll-Hypaque separation. Surface markers on the leukemic cells were: sheep erythrocyte rosette +, OKT3 +, OKT4 +, OKT8 -, Ia -, and surface immunoglobulin -. The leukemic cells were cryopreserved until use.Cryopreserved cells were thawed and 2 x 107 cells in alum were injected intraperitoneally into BALB/c mice (The Jackson Laboratories, Bar Harbor, ME). 3 wk later the mice were boosted with an equal number of cells, and splenic leukocytes were obtained for fusion 3 d later. Spleen cells were fused with SP 2/0 cells by the technique of Kennett et al. (5). Supernatants from viable hybridoma cultures were screened by standard indirect immunofluorescence technique by the use of fluorescein-conjugated Fab~ fragments of goat anti-mouse IgG antiserum (Tago Inc., Burlingame, CA) and a Cytofluorograf 30-H (Ortho Diagnostic Systems Inc., Westwood, MA). Results obtained by flow cytometry were correlated by visual immunofluorescence. Hybridomas of interest were cloned in soft agar. Cloned cells were injected into pristane-primed BALB/c mice and the monoclonal antibody was isolated from the ascites by ammonium sulfate precipitation and ion exchange chromatography.Normal peripheral blood mononuclear cells were subjected to rosetting with neuraminidase-treated sheep erythrocytes. Rosetting and nonrosetting populations plus granulocytes, erythrocytes, and platelets were used to screen the reactivity of the cloned hybridomas. Human T cell lines (MOLT 4, CEM-T, Jurkat, KE 37, 1301, 3639, and HUT 102), B cell lines (8866, Cess, and Josh 7), and myeloid cell lines (K 562, HL 60, and U 937) were also evaluated by flow cytometry. T cell blasts were generated by stimulation with concanavalin A at 10 #g/ml (Sigma Chemical Co., St. Louis, MO), phytohemagglutinin 1:100, or pokeweed mitogen 1:100 (Gibco Laboratories, Grand Island, NY) and stained with the monoclonal antibodies. Samples of peripheral blood from patients with various T cell malignancies were also analyzed by indirect immunofluorescence. The malignancies included two samples of b...
Antibodies directed against the beta chain of the T cell receptor (anti-Vbeta antibodies) are useful to identify the Vbeta repertoire of T cells in various diseases and to quantify numbers of Vbeta-bearing T cells. The goals of this study were to identify Vbeta+ cases of leukemic phase cutaneous T cell lymphoma (CTCL) and to compare the percentage of positive calls with other measures of blood tumor burden, i.e., lymphocyte subsets with a CD4+CD7- and CD4+CD26- phenotype and Sezary cell counts. Thirty-three of 49 (67%) cases of leukemic CTCL reacted with an anti-Vbeta antibody. When combined with reports from the literature, the frequency of Vbeta5 (probably Vbeta5.1) usage was relatively high when compared with Vbeta2 that is also frequently expressed by normal CD4+ T cells. The percentage of Vbeta+ cells correlated to the percentage of CD4+CD7- and CD4+CD26- cells for cases in which the neoplastic cells were deficient in expression of CD7 and CD26, respectively, but not the Sezary cell count. We hypothesize that the increased Vbeta5.1 usage in CTCL may be the result of depletion of Vbeta2 and other Vbeta-bearing T cells by staphylococcal superantigens prior to neoplastic transformation, resulting in a relative increase in the frequency of Vbeta5.1 usage in CTCL.
We studied the role of fine-needle aspiration (FNA) in the evaluation of lymphadenopathy associated with cutaneous T-cell lymphoma (CTCL) in 11 patients with lymphadenopathy and compared findings with corresponding histologic material. Molecular genetic analysis for T-cell clonality by polymerase chain reaction (PCR) was performed on all aspirates. Immunophenotyping was successful in 4 of 7 cases in which flow cytometry was attempted from the aspirated material. Cytologic evaluation of FNA samples correlated strongly with histologic rating of involvement based on numbers of atypical cerebriform lymphocytes in the nodal specimen. Of 7 nodal specimens with scattered or small groups of atypical cells in the background of dermatopathic lymphadenopathy (LN1-2), the cytologic diagnosis was interpreted as reactive in all instances. Of 4 specimens with highly suspect (LN3) or definite histologic involvement (LN4), the cytologic diagnosis was likewise suspect or malignant. The correlation between molecular genetic studies on FNA samples and studies on tissue was not significant; in 2 cases, a T-cell clone was detected in the nodal tissue sample but not in the FNA sample, suggesting undersampling. A T-cell clone was detected by PCR in 5 of 7 nodal specimens judged reactive by FNA biopsy or histologic assessment. FNA for cytologic and molecular genetic analysis is a useful method to evaluate lymphadenopathy associated with CTCL and may obviate the need for surgical biopsy.
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