Volume 123, Issue 4, Pages 1031-1043 (October 2002) Quantitation and phenotypic analysis of natural killer T cells in primary biliary cirrhosis using a human CD1d tetramer  Hiroto Kita, Olga V. Naidenko, Mitchell Kronenberg, Aftab A. Ansari, Paul Rogers, Xiao–Song He, Frits Koning, Toshifumi Mikayama, Judy Van de Water, Ross L. Coppel, Marshall Kaplan, M.Eric Gershwin  Gastroenterology  Volume 123, Issue 4, Pages 1031-1043 (October 2002) DOI: 10.1053/gast.2002.36020 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Flow cytometric analysis of PBMCs from a normal individual. Displayed in the dot plots are cells gated by forward scattering and side scattering. The cells within the box are considered positive. The number next to the box is the percentage of positively stained cells in the lymphocyte population. (A) Cells stained with FITC-labeled anti-CD3 antibody and PE-labeled hCD1d tetramer without α-GalCer. (B) Cells stained with FITC-labeled anti-CD3 antibody and PE-labeled hCD1d tetramer loaded with α-GalCer. (C) Cells stained with FITC-labeled anti-TCRαβ antibody and PE-labeled hCD1d tetramer loaded with α-GalCer. (D) Cells stained with FITC-labeled anti-TCR Vα24 antibody and PE-labeled anti-TCR Vβ11 antibody. (E) Cells stained with FITC-labeled anti-TCR Vα24 antibody and PE-labeled hCD1d tetramer loaded with α-GalCer. Gastroenterology 2002 123, 1031-1043DOI: (10.1053/gast.2002.36020) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Frequencies of TCR Vα24+ Vβ11+ cells and CD3+ tetramer+ cells in peripheral lymphocytes. (A) Healthy individuals (n = 28). (B) PBC patients (n = 13). Gastroenterology 2002 123, 1031-1043DOI: (10.1053/gast.2002.36020) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Enrichment of hCD1d tetramer-reactive cells from a normal individual. CD14 depleted PBMCs were stained with FITC-labeled anti-TCR Vα24 antibody and PE-labeled hCD1d tetramer, followed by incubation with anti-PE microbeads and magnetic separation to enrich tetramer-binding cells. Displayed in the dot plots are cells gated by forward scattering and side scattering. The number next to the box is the percentage of tetramer+ cells in the lymphocyte population. (A) Before enrichment. (B) After enrichment. Gastroenterology 2002 123, 1031-1043DOI: (10.1053/gast.2002.36020) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) Healthy donor PBMCs or (B) PBMCs cultured for 12 days with α-GalCer and recombinant human IL-2 were costained with FITC-labeled anti-TCR Vα24 antibody, PE-labeled anti-TCR Vβ11 antibody, and TC-labeled tetramer. (A) Flow cytometric analysis of fresh PBMCs from a normal individual with tetramer staining on gated PBMCs cell populations; TCR Vα24− Vβ11+ (R1) cells, TCR Vα24+ Vβ11+ (R2) cells, TCR Vα24+ Vβ11− (R3) cells, and TCR Vα24− Vβ11− (R4) cells. (B) Flow cytometric analysis of PBMCs from a normal individual after in vitro stimulation with α-GalCer and recombinant human IL-2 with tetramer staining within TCR Vα24+ Vβ11+ cells (heavy line) and TCR Vα24− Vβ11− cells (thin line) (bottom left); TCR Vα24+ Vβ11+ staining within tetramer+ cells (bottom right). Gastroenterology 2002 123, 1031-1043DOI: (10.1053/gast.2002.36020) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Analysis of TCR Vα24 diversity of CD1d-αGalCer-restricted NKT cells. Fragment length analysis of the Vα 24JαQ CDR3 region is depicted following PCR amplifications with primers for Vα24 and JαQ. The intensity of the fluorescence is represented in arbitrary units as a function of CDR3 length in amino acids. Results from 3 donors are shown. Gastroenterology 2002 123, 1031-1043DOI: (10.1053/gast.2002.36020) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 CD4 and CD8 coreceptor expression by the tetramer-reactive T cells before and after in vitro stimulation. The cells were stained with FITC-labeled anti-CD3 antibody, PE-labeled tetramer, TC-labeled anti-CD4 antibody, and APC-labeled anti-CD8 antibody. Cells gated by forward scattering and side scattering. (A) The frequency of CD4− CD8−, CD4+ CD8−, and CD4− CD8+ subpopulations within the tetramer-reactive population in the PBMC of normal individuals (n = 8). (B) The frequency of the same PBMCs after in vitro stimulation with α-GalCer and recombinant human IL-2 (n = 8). Gastroenterology 2002 123, 1031-1043DOI: (10.1053/gast.2002.36020) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 CD8α CD8β coreceptor expression by tetramer-reactive T cells before and after in vitro stimulation. (A) Flow cytometric analysis of ex vivo PBMCs from a normal individual. Cells stained with FITC-labeled anti-CD8α antibody, PE-labeled anti-CD8β antibody, and TC-labeled hCD1d tetramer loaded with α-GalCer. Displayed in the dot plots are cells gated by forward scattering and side scattering and tetramer positive populations. (B) The frequency of CD8α+ CD8β+ (■) and CD8α+ CD8β− (□) cells in tetramer+ peripheral lymphocytes from normal individuals after in vitro stimulation with α-GalCer and recombinant human IL-2 (n = 8). Gastroenterology 2002 123, 1031-1043DOI: (10.1053/gast.2002.36020) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 The frequency of CD56+ and CD161+ cells in tetramer+ peripheral lymphocytes of normal individuals (n = 11). Gastroenterology 2002 123, 1031-1043DOI: (10.1053/gast.2002.36020) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 The frequency of hCD1d tetramer+ Vα24+ cells before and after enrichment in healthy donors vs. PBC patients. (A) Healthy donors (n = 6). (B) PBC patients (n = 6). Gastroenterology 2002 123, 1031-1043DOI: (10.1053/gast.2002.36020) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 10 The frequency of TCR Vα24+ Vβ11+ cells and hCD1d tetramer+ CD3+ cells in LILs. (A) Normal livers (n = 4). (B) PBC livers (n = 12). Gastroenterology 2002 123, 1031-1043DOI: (10.1053/gast.2002.36020) Copyright © 2002 American Gastroenterological Association Terms and Conditions