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SPECIFIC IgG ANTIBODY SECRETION FROM HLA TETRAMER ISOLATED B CELLS.
Dessislava Kopchaliiska PhD 1, Mary S. Leffell PhD 1 and Andrea A. Zachary PhD 1. 1 Medicine, Johns Hopkins University, Baltimore, MD, USA .
MHC tetramer staining allows direct detection of antigen-specific T cells. We have shown previously that class I tetramers (tet) can be used for detection of allo-reactive B cells in sensitized renal transplant patients. Here we report detection of HLA-specific antibody (Ab) produced by tet-bound B cells in culture. Methods: Mononuclear spleen cells isolated from a patient with known sensitization to HLA B7 and A24 were incubated with PE-conjugated HLA-B*0702 and HLA-A*2402 tetramers. The frequencies of tet+ cells were: A24 - 6.6%; B7 - 8.9%. PE-tet+ cells were isolated using EasySep
PE Selection magnetic nanoparticles (StemCell Technologies Inc., Canada). Isolated cells were cultured in RPMI 1640 with 10% fetal calf serum and supplemented with co-stimulatory factors as follows:1) CpG (10
l/ml), CD40L (1/250 dil), IL-2 (10ng/ml), IL-10 (10ng/ml); 2) CD40L, IL-2, IL-10; 3) CpG only; and 4) medium only. After culture for 6 days at 37oC with 5% CO2, IL-2 and IL-10 were added to cultures 1 and 2 for 5 additional days. Supernatants were collected and tested for specific antibody secretion with LIFECODES Class I ID (Tepnel, Stamford, CT). Results: IgG Ab specific for A24 or B7 was detected in two of the four respective cultures of isolated A24 or B7 tet+ cells. The highest levels of specific antibody were found in cultures stimulated with CpG, CD40L, IL-2 and IL-10, followed by cultures with CD40L, IL-2, and IL-10. No HLA specific antibody was detectable in cultures supplemented with only CpG or medium. Conclusions: These results confirm the specificity of tetramer binding through surface Ig receptors on B cells. Further, tetramer labeled cells can be isolated and induced with appropriate co-stimulation to secrete HLA specific Ab in culture.