Cell-microarray analysis of antigen-specific B-cells: Single cell analysis of antigen receptor expression and specificity

2007 ◽  
Vol 71A (11) ◽  
pp. 961-967 ◽  
Author(s):  
Kazuto Tajiri ◽  
Hiroyuki Kishi ◽  
Yoshiharu Tokimitsu ◽  
Sachiko Kondo ◽  
Tatsuhiko Ozawa ◽  
...  
Keyword(s):  
B Cells ◽  
Single Cell ◽  
Microarray Analysis ◽  
Single Cell Analysis ◽  
Antigen Receptor ◽  
Receptor Expression ◽  
Cell Analysis ◽  
Cell Microarray

Densely-Packed Microbowl Array with Balanced Dielectrophoretic Forces for Single-Cell Microarray

2009 ◽  
Vol 1222 ◽  
Author(s):  
Maesoon Im ◽  
Dong-Haan Kim ◽  
Joo-Hyung Lee ◽  
Jun-Bo Yoon ◽  
Yang-Kyu Choi
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Three Dimensional ◽  
Average Diameter ◽  
Experimental Result ◽  
Cell Analysis ◽  
Large Area ◽  
Latex Beads ◽  
Nickel Electroplating ◽  
Cell Microarray

AbstractIn this paper, we demonstrate a perfectly-ordered microbowl array with balanced dielectrophoresis (DEP) for a high-throughput single-cell analysis. In order to fabricate well-ordered microbowl array in a large area, we utilized three-dimensional diffuser lithography for photoresist mold and nickel electroplating technique for final microbowl structures on a silicon substrate. Single microbowl has six sharp apexes surrounding the microbowl perimeter. Each microbowl has a diameter of 10 μm, and a height of 9 μm, which can be controllable by patterns on mask and lithography conditions. To investigate feasibility for application to the microbowl array as a single-cell microarray, we used latex beads of 6.4 μm in an average diameter to be captured by dielectrophoretic force. The nickel microbowl array densely packed with a hexagonal geometry played as a bottom electrode, and an ITO-coated glass covered the nickel microbowl array as a top electrode while keeping a uniform gap between two electrodes. After injecting solution containing latex beads through the gap, we applied an AC signal (2 VPP, 1 MHz) between two electrodes to induce high electric field near the sharp apexes of the single microbowl. A negative DEP trap is formed at the center of the single microbowl with balanced DEP force from the six apexes. The experimental result shows that injected latex beads had been successfully and uniformly aligned and trapped at the microbowl array sustained by negative DEP.

Single-Cell Analysis of Costimulation by B Cells, Endothelial Cells, and Fibroblasts Demonstrates Heterogeneity in Responses of CD4+ Memory T Cells

1999 ◽  
Vol 194 (2) ◽  
pp. 150-161 ◽  
Author(s):  
Lisa L. Salazar Murphy ◽  
Melissa M. Mazanet ◽  
Angela C. Taylor ◽  
Javier Mestas ◽  
Christopher C.W. Hughes
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
B Cells ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Memory T Cells ◽  
Cell Analysis

Single-Cell Analysis of FVIII-Specific Memory B Cells in Murine Hemophilia A.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1157-1157 ◽  
Author(s):  
Christina Hausl ◽  
Rafi U. Ahmad ◽  
Bernhard Baumgartner ◽  
Hans Peter Schwarz ◽  
Hartmut Ehrlich ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Cd4 T Cells ◽  
Single Cell Analysis ◽  
Memory B Cells ◽  
Cell Analysis ◽  
Memory B Cell ◽  
Specific Memory

Abstract The elimination of FVIII-specific memory B cells is an essential step in the design of new therapeutic strategies for the induction of immune tolerance in hemophilia A with FVIII inhibitors. Using a mouse model of hemophilia A we recently reported that low dose FVIII stimulates the differentiation of FVIII-specific memory B cells into antibody-secreting plasma cells whereas high dose FVIII inhibits this process. The inhibition of memory-B-cell re-stimulation is irreversible and seems to be due to an induction of apoptosis. Further understanding of the complex interactions that lead to either re-stimulation and differentiation of memory B cells or inhibition and eradication of these cells requires appropriate technologies for single-cell analysis and functional studies. We established a new technology for single-cell analysis and cell sorting of FVIII-specific murine memory B cells. A combination of magnetic bead separation and multi-color flow cytometry enabled us to analyze and purify FVIII-specific memory B cells obtained from hemophilic mice treated with FVIII. In a first step, we depleted undesirable cell populations (IgM+, IgD+, CD11c+, F4/80+, Gr1+ and CD49b+ cells) from total spleen cells by magnetic bead separation. In a second step, we used multicolor flow cytometry to exclude CD4+ T cells and analyze the FVIII-specific memory B cell compartment. This compartment was specified by staining the specific B-cell receptor with FVIII and anti-IgG antibodies. Frequencies of cells in this compartment ranged from 0.1–0.5% of total spleen cells in animals treated with 4 intravenous doses of FVIII, given at weekly intervals. We could not detect any FVIII-specific memory B cells in naïve mice. By means of single cell sorting we isolated FVIII-specific memory B cells for further functional studies. We were able to cultivate FVIII-specific memory B cells in microwell cultures in vitro and differentiate them into antibody-secreting plasma cells. The re-stimulation and differentiation of single-cell sorted memory B cells was strictly dependent on the presence of activated CD4+ T cells. CD4+ T cells obtained from naïve mice did not support the memory response. Furthermore, the re-stimulation and differentiation of memory B cells in the presence of activated CD4+ T cells did not require additional dendritic cells for antigen presentation. Obviously, memory B cells provide sufficient antigen presentation to CD4+ T cells to enable them to trigger the memory response. Our approach for single-cell analysis and purification of FVIII-specific memory B cells provides a new tool for tracking memory B cell populations in vivo and for directly analyzing the regulation of memory B cell function. It opens the field for future studies which should elucidate signals and molecules involved in activation or inhibition and eradication of FVIII-specific memory B cells. These activities will eventually lead to the identification of targets for the design of new treatment strategies for patients with FVIII inhibitors.

scholarly journals Single-cell analysis of germinal-center B cells informs on lymphoma cell of origin and outcome

2020 ◽  
Vol 217 (10) ◽  
Author(s):  
Antony B. Holmes ◽  
Clarissa Corinaldesi ◽  
Qiong Shen ◽  
Rahul Kumar ◽  
Nicolo Compagno ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Plasma Cells ◽  
Single Cell Analysis ◽  
Affinity Maturation ◽  
Cell Analysis ◽  
Cell Of Origin ◽  
B Cell Lymphomas ◽  
Cell Affinity

In response to T cell–dependent antigens, mature B cells are stimulated to form germinal centers (GCs), the sites of B cell affinity maturation and the cell of origin (COO) of most B cell lymphomas. To explore the dynamics of GC B cell development beyond the known dark zone and light zone compartments, we performed single-cell (sc) transcriptomic analysis on human GC B cells and identified multiple functionally linked subpopulations, including the distinct precursors of memory B cells and plasma cells. The gene expression signatures associated with these GC subpopulations were effective in providing a sc-COO for ∼80% of diffuse large B cell lymphomas (DLBCLs) and identified novel prognostic subgroups of DLBCL.

Clonotypic IgM V/D/J sequence analysis in Waldenstrom macroglobulinemia suggests an unusual B-cell origin and an expansion of polyclonal B cells in peripheral blood

Blood ◽  
2004 ◽  
Vol 104 (7) ◽  
pp. 2134-2142 ◽  
Author(s):  
Jitra Kriangkum ◽  
Brian J. Taylor ◽  
Steven P. Treon ◽  
Michael J. Mant ◽  
Andrew R. Belch ◽  
...  
Keyword(s):  
B Cells ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Gene Families ◽  
Immunoglobulin M ◽  
Cell Analysis ◽  
Fragment Analysis ◽  
Variable Regions ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia

Abstract Analysis of clonotypic immunoglobulin M (IgM) from 15 patients with Waldenstrom macroglobulinemia (WM) showed a strong preferential use of the VH3/JH4 gene families. Identification of the WM IgM V/D/J was validated using single-cell analysis, confirming its presence in most B cells. Despite the extensive hypermutated VH genes in 13 of 15 patients, statistical analysis of framework/complementary-determining region (FR/CDR) mutation patterns suggests that they might have escaped antigenic selection. Neither intraclonal diversity nor isotype switching was detectable. Membranous and secreted forms of clonotypic IgM transcripts were present in bone marrow and blood. Single-cell analysis showed that clonotypic B cells coexpress CD20, surface IgM (sIgM), and sIgD but that they lack CD138. Most B cells lacked memory marker CD27 despite their hypermutated variable regions otherwise suggestive of memory status. At diagnosis, circulating B cells in WM are largely clonotypic. However, when monoclonal IgM levels are decreased, clonotypic frequencies are substantially reduced despite elevated CD20+ cells, shown to be polyclonal by DNA sequencing and CDR3 fragment analysis. Thus, WM includes the expansion of circulating, polyclonal B cells. Overall, this work suggests that WM may originate from a largely VH3-restricted, somatically mutated, predominantly CD27-IgM+IgD+ population that cannot undergo class switching, suggestive of B cells that might have bypassed the germinal center. (Blood. 2004;104:2134-2142)

scholarly journals 331 Single cell analysis reveals diversity of phenotype and function of autoantigen-specific B cells in systemic sclerosis

2017 ◽  
Vol 137 (10) ◽  
pp. S249
Author(s):  
A. Yoshizaki ◽  
T. Fukasawa ◽  
S. Ebata ◽  
Y. Asano ◽  
K. Mawatari ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
B Cells ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Cell Analysis ◽  
And Function
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