1. Detection of Antigen-Specific B Cells in Patients with Pemphigus Vulgaris by Enzyme- Linked Immunospot Assay: Requirement of T Cell Collaboration for Autoantibody Production 
Koji Nishifuji, Masayuki Amagai, Masataka Kuwana, Toshiro Iwasaki, Takeji Nishikawa 
Journal of Investigative Dermatology 
Volume 114, Issue 1, Pages (January 2000)
DOI: /j x
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Quantitative detection of anti-Dsg3 mouse hybridoma cells by ELISPOT assay. (A) 5H10 cells were incubated on rDsg3-coated or BSA-coated PVDF-bottomed 96-well multititer plates, then bound IgG was detected by alkaline phosphatase-conjugated anti-mouse IgG antibody. Clear spots were seen on rDsg3-coated plates, but not on BSA-coated plates. (B) Various numbers of 12A2 cells were incubated on rDsg3-coated plates with and without negative control 16H11 cells. The number of spots represented very well the number of the 12A2 cells seeded when 105 or less 16H11 cells coexisted.
Journal of Investigative Dermatology  , 88-94DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Detection of anti-Dsg3 IgG-secreting B cells in different tissues of Dsg3-immunized mice. BALB/C mice were immunized with rDsg3 with complete and incomplete Freund’s adjuvant, and mononuclear cells were isolated from spleen, bone marrow, mesenteric and inguinal lymph nodes, and peripheral blood. Fresh mononuclear cells were serially diluted and incubated on rDsg3-coated plates for 4 h. Anti-Dsg3 IgG-secreting B cells were detected mostly in spleen and bone marrow.
Journal of Investigative Dermatology  , 88-94DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Frequency of anti-Dsg3 IgG-producing B cells and Dsg3-specific memory B cells in PBMC of patients with PV. (A) Fresh PBMC from patients or normal individuals were directly applied to ELISPOT assay. (B) PBMC from patients or normal individuals were cultured in vitro with PWM and rDsg3 for 4 d, and then subjected to ELISPOT-based limiting dilution assay. Frequency was obtained as number of spots per 105 PBMC and is shown in semilog scale. Patients were subdivided according to disease activity (scales, 0–3). The sensitivity of this assay was 0.5 positive cells per 105 PBMC.
Journal of Investigative Dermatology  , 88-94DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Effects on in vitro antibody production by depletion of CD4 + cells. The number of spots were compared between PBMC with and without depletion of CD4 + cells. PBMC was treated with anti-CD4 MoAb coupled magnetic beads followed by magnetic removal of bead-bound cells. Non-depleted control was prepared simultaneously without addition of the magnetic beads. Then, the cells were cultured for 7 d with determined concentration of PWM and rDsg3. The number of spots were significantly reduced by depletion of CD4 + cells.
Journal of Investigative Dermatology  , 88-94DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Effect of anti-MHC class II MoAb on in vitro proliferation of B cells. Anti-HLA-DR, DQ, and DP MoAb and isotype control antibodies were added in the beginning of culture and subjected to ELISPOT assay. Anti-HLA-DR (in patients KH and ET) and anti-HLA-DQ (in patients KH, ET, and CK) MoAb markedly suppressed the number of the spots whereas anti-HLA-DP and isotype controls had no apparent effect.
Journal of Investigative Dermatology  , 88-94DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions