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Tissue samples were fixed in 10% buffered formalin. Sections (5 μm) were stained with hematoxylin and eosin (H&E). Immunophenotyping was done with an avidin-biotin immunoperoxidase technique. Anti-CD3 (purified rabbit serum, 1:1000; Dako), and anti-B220 (rat monoclonal, 1:1000; PharMingen) were incubated overnight at 4°C. Samples were later incubated with biotinylated secondary antibodies (Vector Labs) for 30 min (goat antibody to rabbit IgG, 1:100; rabbit antibody to rat IgG, 1:100), and then with avidin-biotin peroxidase (1:25 dilution, Vector Labs) for 30 min. Diaminobenzadine was used as the chromogen and hematoxylin was used as the counterstain.
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Single-cell suspensions of spleen and lymph nodes were depleted of mature red blood cells by hypotonic lysis and were stained with the following conjugated antibodies: anti-CD3ε, anti-B220, anti-Thyl, anti-CD4, anti-CD8a (lineage markers); anti-CD5, anti-CD95, anti-CD54, anti-CD69, anti-CD44, anti-B7-2 (activation markers). All antibodies were from PharMingen. Flow cytometry was done with a FAC-Scan (Becton Dickinson) and the data were analyzed with CELLQuest software (Becton Dickinson).
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We thank J. L. Zhou for help with the ELISA, M. Jiao for preparation of pathological samples, and M. De Acetis for help with protein immunoblots and mice. P.P.P. is a Scholar of the Leukemia Society of America. Supported by National Cancer Institute grants CA-08748 and CA-82328 and the I. T. Hirschl/M. Weill-Caulier Foundation (P.P.P.), and by NIH grant AR45482 and the Systemic Lupus Ezythemetosus Foundation, New York (K.B.E.).
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