Vaccinia virus–specific memory B cells are enriched in the spleen and decreased in blood after splenectomy or rituximab treatment. ELISPOT assays were performed after 6 days of in vitro polyclonal stimulation of mononucleated cells. (A) ELISPOT showing a 10-fold enrichment of antivaccinia memory B cells in the spleen. MVA, anti-IgG, or an unrelated antigen (KLH) were used as coating antigens. Blood and spleen samples from patient V10 (Table 1). Shown is one representative ELISPOT well of 3, corresponding to the indicated dilution for each antigen. (B) Frequency of anti–vaccinia virus memory B cells in blood and spleen, determined by ELISPOT assay. The insert shows data from 3 patients (V10, V13, V18, Table 1) for whom both blood and spleen were analyzed. (C) Frequency of anti–vaccinia virus memory B cells determined by ELISPOT assay in blood of splenectomized (SPL) and rituximab-treated (RTX) patients, compared with healthy donors (control, same samples as in B). Bold lines indicate the median values for groups with sample size more than 5. Dashed symbols correspond to maximum estimates for individuals for whom specific memory B cells were undetectable (“Memory B-cell assay”). P values are determined by a nonparametric Kruskal-Wallis 2-sided test.