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Tracking alloreactive B cells with HLA tetramers
S130
Abstracts
5.07 #167
TRACKING ALLOREACTIVE B CELLS WITH HLA TETRAMERS Mary S. Leffell, Dessislava Kopchaliiska, Andrea A. Zachary. Medicine, Johns Hopkins University School of Medicine, Baltimore, MD MHC tetramers afford powerful analysis of T lymphocytes. Tetramers should also bind to HLA-specific surface immunoglobulin receptors on B cells. We developed a procedure to label B cells with HLA tetramers and tested peripheral blood samples from 16 patients with known anti-HLA antibodies. B cells were enriched by negative depletion of T cells with anti-CD2 magnetic beads or by positive selection with anti-CD19 beads. 5⫻106 enriched B cells were stained with 10 of HLA-A0201MART1-PE, HLA-A0201GAG-PE, and/or HLA-B0702p24-APC(Beckman Coulter) at 4 degrees for 45 minutes. Cells were also labeled with PE or FITC labeled anti-CD3 and CD19 (BD Biosciences/ Pharmingen). After washing and fixation, 2 or 3 color analysis was performed on a minimum of 2⫻104 total lymphocytes and 1⫻104 CD19⫹ cells using a FACSCalibur cytometer and Cell Quest software (Becton Dickinson). Controls included non-sensitized males and other patients with either A2 or B7 in their phenotype. The mean frequency of CD19⫹A0201tetramer⫹ cells for 10 patients with known A2 sensitization was significantly higher than that of 8 controls: 7.4 ⫹ 6.8% v.s. 1.2 ⫹ 0.6%, P⫽0.013. The frequency of binding to non-CD19 cells ranged from 0.3 to 1.0% among both patients and controls. A0201 tetramer frequencies were higher among patients with current v.s. those with historic anti-A2 antibodies (11.8 ⫹ 7.6 and 3.1 ⫹ 0.4, respectively). The mean frequency of CD19⫹B0702⫹ cells from 3 patients with historic B7 sensitization was 3.31%, comparable to that observed with historic antiA2 sensitization. Serial samples from one patient whose anti-A2 levels changed from undetectable by ELISA to a PRA⫽96 gave respective CD19⫹A0201⫹ frequencies of 1.4 and 15.9%. These data indicate that MHC tetramers provide a sensitive and specific means to assess allo-reactive B lymphocyte frequencies. This work was supported, in part, by funding from MedImmune, Inc.
5.07 #168
DIFFERENTIAL EXPRESSION OF HLA CLASS I ON B CELL SUBSETS Howard M. Gebel,1 Peter Nickerson,2 Martin Shelton,1 Thomas M. Ellis,3 Robert A. Bray.1 1Pathology, Emory University Hospital, Atlanta, GA; 2Medicine, University of Manitoba, Winnipeg, MB, Canada; 3Histocompatibility and Immunogenetics Laboratory, Blood Center of Southeastern Wisconsin, Milwaukee, WI The interpretation of a B-cell⫹/T-cell- flow cytometric crossmatch(FCXM) can be problematic. B cell⫹ FCXMs could be due to antibodies against, class II alone, class I alone (low titer), class I and II (low titer), or non-HLA antigens. Additionally, when B cells are incubated with a negative control serum and stained with anti-IgG, two populations of cells (bright and dim) are observed. While these two populations may reflect nonspecific binding of IgG to Fc receptors, both populations remain after pronase treatment. The bright population (5–20% of B cells) expresses surface IgG (sIgG⫹), while the dim population (80 –95% of B cells) lacks surface IgG (sIgG-). A B cell FCXM has been interpreted as positive when the fluorescence cutoff value is exceeded and the two B cell populations merge together (ie; single peak). This is predicated on the assumption that class I and class II are equally expressed on both B cell populations. To test this theory, we used threecolor flow cytometry to assess MHC expression on sIgG⫹ and sIgG- B cells. Briefly, peripheral blood (n⫽10) and lymph node (n⫽11) cells were co-stained with CD20 PerCP, anti-IgG FITC and either anti-class I (W632) or class II (L243)-PE. While the expression of HLA class II was identical on all examples of sIgG⫹ and sIgG- B cells, the expression of class I on sIgG⫹ and sIgG- B cells was distinctly different. The sIgG⫹ B cells displayed a unimodal peak with a median fluorescence (MRF) of ⬃3000 while class I expression on sIgG- B cells was bimodal ( 75% of cells with a MRF of ⬃1000 and 5% with a MRF of ⬃3000). Clinically, these studies indicate that HLA class I antibodies must still be considered when the cutoff value is exceeded but the architecture is identical to that of the negative control.
Abstracts
5.07 #167
TRACKING ALLOREACTIVE B CELLS WITH HLA TETRAMERS Mary S. Leffell, Dessislava Kopchaliiska, Andrea A. Zachary. Medicine, Johns Hopkins University School of Medicine, Baltimore, MD MHC tetramers afford powerful analysis of T lymphocytes. Tetramers should also bind to HLA-specific surface immunoglobulin receptors on B cells. We developed a procedure to label B cells with HLA tetramers and tested peripheral blood samples from 16 patients with known anti-HLA antibodies. B cells were enriched by negative depletion of T cells with anti-CD2 magnetic beads or by positive selection with anti-CD19 beads. 5⫻106 enriched B cells were stained with 10 of HLA-A0201MART1-PE, HLA-A0201GAG-PE, and/or HLA-B0702p24-APC(Beckman Coulter) at 4 degrees for 45 minutes. Cells were also labeled with PE or FITC labeled anti-CD3 and CD19 (BD Biosciences/ Pharmingen). After washing and fixation, 2 or 3 color analysis was performed on a minimum of 2⫻104 total lymphocytes and 1⫻104 CD19⫹ cells using a FACSCalibur cytometer and Cell Quest software (Becton Dickinson). Controls included non-sensitized males and other patients with either A2 or B7 in their phenotype. The mean frequency of CD19⫹A0201tetramer⫹ cells for 10 patients with known A2 sensitization was significantly higher than that of 8 controls: 7.4 ⫹ 6.8% v.s. 1.2 ⫹ 0.6%, P⫽0.013. The frequency of binding to non-CD19 cells ranged from 0.3 to 1.0% among both patients and controls. A0201 tetramer frequencies were higher among patients with current v.s. those with historic anti-A2 antibodies (11.8 ⫹ 7.6 and 3.1 ⫹ 0.4, respectively). The mean frequency of CD19⫹B0702⫹ cells from 3 patients with historic B7 sensitization was 3.31%, comparable to that observed with historic antiA2 sensitization. Serial samples from one patient whose anti-A2 levels changed from undetectable by ELISA to a PRA⫽96 gave respective CD19⫹A0201⫹ frequencies of 1.4 and 15.9%. These data indicate that MHC tetramers provide a sensitive and specific means to assess allo-reactive B lymphocyte frequencies. This work was supported, in part, by funding from MedImmune, Inc.
5.07 #168
DIFFERENTIAL EXPRESSION OF HLA CLASS I ON B CELL SUBSETS Howard M. Gebel,1 Peter Nickerson,2 Martin Shelton,1 Thomas M. Ellis,3 Robert A. Bray.1 1Pathology, Emory University Hospital, Atlanta, GA; 2Medicine, University of Manitoba, Winnipeg, MB, Canada; 3Histocompatibility and Immunogenetics Laboratory, Blood Center of Southeastern Wisconsin, Milwaukee, WI The interpretation of a B-cell⫹/T-cell- flow cytometric crossmatch(FCXM) can be problematic. B cell⫹ FCXMs could be due to antibodies against, class II alone, class I alone (low titer), class I and II (low titer), or non-HLA antigens. Additionally, when B cells are incubated with a negative control serum and stained with anti-IgG, two populations of cells (bright and dim) are observed. While these two populations may reflect nonspecific binding of IgG to Fc receptors, both populations remain after pronase treatment. The bright population (5–20% of B cells) expresses surface IgG (sIgG⫹), while the dim population (80 –95% of B cells) lacks surface IgG (sIgG-). A B cell FCXM has been interpreted as positive when the fluorescence cutoff value is exceeded and the two B cell populations merge together (ie; single peak). This is predicated on the assumption that class I and class II are equally expressed on both B cell populations. To test this theory, we used threecolor flow cytometry to assess MHC expression on sIgG⫹ and sIgG- B cells. Briefly, peripheral blood (n⫽10) and lymph node (n⫽11) cells were co-stained with CD20 PerCP, anti-IgG FITC and either anti-class I (W632) or class II (L243)-PE. While the expression of HLA class II was identical on all examples of sIgG⫹ and sIgG- B cells, the expression of class I on sIgG⫹ and sIgG- B cells was distinctly different. The sIgG⫹ B cells displayed a unimodal peak with a median fluorescence (MRF) of ⬃3000 while class I expression on sIgG- B cells was bimodal ( 75% of cells with a MRF of ⬃1000 and 5% with a MRF of ⬃3000). Clinically, these studies indicate that HLA class I antibodies must still be considered when the cutoff value is exceeded but the architecture is identical to that of the negative control.