Lineages of human T-cell clones, including T helper 17/T helper 1 cells, isolated at different stages of anti–factor VIII immune responses by Ruth A. Ettinger, Eddie A. James, William W. Kwok, Arthur R. Thompson, and Kathleen P. Pratt Blood Volume 114(7):1423-1428 August 13, 2009 ©2009 by American Society of Hematology

Proliferation and cytokine secretion of T-cell clones isolated from subject 17A 19 weeks and 21 months after inhibitor development and from subject 32A at one time point. Proliferation and cytokine secretion of T-cell clones isolated from subject 17A 19 weeks and 21 months after inhibitor development and from subject 32A at one time point. Resting T-cell clones were stimulated with FVIII2194-2213 (0.1, 1.0, and 10 μM) presented on irradiated PBMCs from an unrelated DRB1*0101 donor. Proliferation (n = 3) was measured by addition of [3H]thymidine at 48 hours, and cells were harvested 18 hours later (A). Cell supernatants were collected after 48 hours to measure IFN-γ (B), IL-4 (C), and IL-17 (D) by ELISA. The proliferation and cytokine levels of cells stimulated with buffer as a negative control were subtracted. Results are presented as stacked bar graphs. (E-F) The total amounts of cytokines secreted after stimulation with FVIII2194-2213 at the 3 concentrations indicated in panel A were summed, and ratios of these total levels were calculated. In all panels, clones are grouped according to their cytokine secretion profiles. Ruth A. Ettinger et al. Blood 2009;114:1423-1428 ©2009 by American Society of Hematology

Cytokine secretion profiles for early lots of IL-17–secreting clones. Cytokine secretion profiles for early lots of IL-17–secreting clones. Resting T-cell clones (first seed lot for 17A-19WK-11 and second seed lots for 17A-19WK-22 and -33) were stimulated with FVIII2194-2213 (0.1, 1.0, and 10 μM) presented on irradiated PBMCs from an unrelated DRB1*0101 donor. Cell supernatants were collected from triplicate wells after 48 hours to measure IFN-γ, IL-4, and IL-17 by ELISA. The cytokine levels of cells stimulated with buffer as a negative control were subtracted. Ruth A. Ettinger et al. Blood 2009;114:1423-1428 ©2009 by American Society of Hematology

Transcription factor mRNA and chemokine receptor expression for FVIII-specific T-cell clones. Transcription factor mRNA and chemokine receptor expression for FVIII-specific T-cell clones. (A) TBX21, GATA3, and RORC mRNA expression in T-cell clones was quantified by real-time quantitative polymerase chain reaction using TaqMan gene expression assays. Expression levels were normalized to 18S rRNA expression. Mean values are shown for TBX21, GATA3, and RORC mRNAs, plotted as stacked bar graphs. Cycle threshold values were determined in triplicate for 2 to 3 samples of each clone. The samples were generated by separate expansions, except for T-cell clones 17A-19WK-10, -22, -33, 17A-21MO-12, and 32A-21, which were evaluated only once because they expanded poorly compared with the other clones. (B) CXCR3, CCR4, and CCR6 cell surface expression on T-cell clones was quantified by flow cytometry. The MFI indicating the specific antibody staining minus the isotype staining was calculated. Mean values from 2 to 3 independent experiments are shown, plotted as stacked bar graphs. Clones are grouped according to their cytokine secretion profiles. One example of the chemokine receptor-staining data is shown in the inset as a conventional histogram overlay (chemokine receptor antibodies, filled histogram; isotype control antibodies, open histogram) with fluorescent intensity plotted on the x-axis (logarithmic scale from 100 to 103 fluorescent units) and cell counts plotted on the y-axis, normalized as percentage of maximum (linear scale from 0%-100%). * indicates that the inset corresponds to clone 17A-19WK-22. Ruth A. Ettinger et al. Blood 2009;114:1423-1428 ©2009 by American Society of Hematology