Enrichment and detection of live antigen-specific CD4(+) and CD8(+) T cells based on cytokine secretion.

Following appropriate antigen-specific stimulation, CD4(+) and CD8(+) T lymphocytes rapidly express cytokines. Based on this stimulation-induced cytokine secretion and using cell surface affinity matrix technology we have developed a new method that permits specific, rapid and efficient detection, isolation and characterization of live antigen-specific CD4(+) and CD8(+) T lymphocytes. The power of this technique is demonstrated here for HLA-A0201-restricted influenza matrix protein peptide 58-66-specific CD8(+) cytotoxic T lymphocytes, influenza A virus- and recombinant tetanus toxin C fragment-specific Th1 cells and tetanus toxoid-specific Th2 cells.

Correlation analysis between frequencies of circulating antigen-specific IgG-bearing memory B cells and serum titers of antigen-specific IgG.

Recent studies in mice have indicated that the long-lasting specific antibody responses seen after vaccination are probably due to the existence of long-lived plasma cells. Therefore, because the maintenance of humoral immunity does not necessarily reflect continuous restimulation of long-lived memory B cells, the question arises as to what degree antibody immunity, as determined by measuring serum immunoglobulin titers against a particular antigen, and memory B cell immunity, as determined by counting circulating memory B cells with specificity for that same antigen, correlate. Here, using a new assay combining two-step immunomagnetic enrichment with multiparameter flow cytometry to detect, enumerate and characterize antigen-specific memory B cells, we show for tetanus toxin C-fragment in blood of normal tetanus toxoid vaccinized donors, and for wasp venom phospholipase A1B in blood of wasp venom-allergic donors undergoing an immune therapy with wasp venom, that there is no statistically significant linear correlation between the frequencies of circulating antigen-specific IgG-bearing memory B cells and the serum titers of antigen-specific IgG. This lack of a statistically significant linear correlation is in accordance with the idea that B memory cells and plasma cells represent independently controlled forms of immunological memory.