A rheostatic mechanism for T-cell inhibition based on elevation of activation thresholds by Jacob Rachmilewitz, Gregory J. Riely, Jui-Han Huang, Aoshuang Chen, and Mark L. Tykocinski Blood Volume 98(13):3727-3732 December 15, 2001 ©2001 by American Society of Hematology

The inhibitory activity of AF, but not CSA, is inversely correlated to stimulus and is overridden by increasing receptor engagement.Jurkat cells were stimulated with various concentrations of PHA in the absence (▪) or presence of either AF (25% ■) or PP14-d... The inhibitory activity of AF, but not CSA, is inversely correlated to stimulus and is overridden by increasing receptor engagement.Jurkat cells were stimulated with various concentrations of PHA in the absence (▪) or presence of either AF (25% ■) or PP14-depleted AF (▴) (A); or in the presence of CSA (100 pg/mL [■] or 2 ng/mL [▵]) (B). IL-2 secretion was determined in the conditioned medium. The data represent the mean of triplicate samples. Comparable results were obtained in 4 separate experiments. (C) Change in absolute IL-2 levels as a function of PHA concentration in the presence of either AF (25% ▪) or CSA (100 pg/mL [■] or 2 ng/mL [▵]), as calculated from the data in panels A and B. Jacob Rachmilewitz et al. Blood 2001;98:3727-3732 ©2001 by American Society of Hematology

AF elevates the threshold number of TCRs required for IL-2 secretion AF elevates the threshold number of TCRs required for IL-2 secretion.Either Jurkat cells or T cells were stimulated with various concentrations of PHA or solid-phase OKT3 in the absence (▪) or presence of AF (○). AF elevates the threshold number of TCRs required for IL-2 secretion.Either Jurkat cells or T cells were stimulated with various concentrations of PHA or solid-phase OKT3 in the absence (▪) or presence of AF (○). (A) Percentage of TCR down-modulation in Jurkat cells as a function of PHA concentration, as measured by CD3 immunofluorescence and flow cytometry. (B) Percentage of TCR down-modulation in T cells as a function of OKT3 concentrations. (C) IL-2 secretion by Jurkat cells as a function of the percentage of TCR down-modulation, as calculated from the data in Figure 1, panel A, and panel A herein. The data represent the mean of triplicate samples. Comparable results were obtained in 3 separate experiments. Jacob Rachmilewitz et al. Blood 2001;98:3727-3732 ©2001 by American Society of Hematology

Influence of absolute numbers of surface TCRs and costimulation on sensitivity to inhibition by AF.(A) Absolute numbers of TCRs determine sensitivity to inhibition by AF. Jurkat cells expressing low or high levels of TCRs were preparatively sorted and stimu... Influence of absolute numbers of surface TCRs and costimulation on sensitivity to inhibition by AF.(A) Absolute numbers of TCRs determine sensitivity to inhibition by AF. Jurkat cells expressing low or high levels of TCRs were preparatively sorted and stimulated with solid-phase OKT3 and anti-CD28 mAbs in the presence or absence of AF (25%). Histograms on the right show the relative CD3 immunostaining of the sorted cell populations. Comparable results were obtained in 2 repeat experiments in which cell populations were sorted independently. (B) Costimulation lowers the threshold number of TCRs required for IL-2 production and renders cells less sensitive to inhibition by AF. Jurkat cells were stimulated with varying concentrations of PHA, with (▵) or without (▪) addition of B7-1–transfected CHO cells. Results in panels A and B are presented as a percentage of inhibition of IL-2 secretion. Jacob Rachmilewitz et al. Blood 2001;98:3727-3732 ©2001 by American Society of Hematology

Hierarchical inhibition by AF of IFN-γ and IL-2 production in peripheral blood T cells.Peripheral blood T cells were stimulated in the presence of fixed activated monocytes, with increasing PHA concentrations, with or without AF or PP14-depleted AF at the i... Hierarchical inhibition by AF of IFN-γ and IL-2 production in peripheral blood T cells.Peripheral blood T cells were stimulated in the presence of fixed activated monocytes, with increasing PHA concentrations, with or without AF or PP14-depleted AF at the indicated concentrations. Conditioned media were collected 48 hours later, and IL-2 (upper panel) and IFN-γ (lower panel) secretions were determined by ELISA. Comparable results were obtained in 4 separate experiments. Jacob Rachmilewitz et al. Blood 2001;98:3727-3732 ©2001 by American Society of Hematology

PP14. Fcγ1 increases OKT3 dose required for T-cell activation PP14.Fcγ1 increases OKT3 dose required for T-cell activation.Purified T cells were stimulated with increasing amounts of solid-phase OKT3 mAbs either alone (♦), in conjunction with anti-CD28 mAb (▪), in conjunction with anti-CD28 mAb plus AF (12.5% vol/vol;... PP14.Fcγ1 increases OKT3 dose required for T-cell activation.Purified T cells were stimulated with increasing amounts of solid-phase OKT3 mAbs either alone (♦), in conjunction with anti-CD28 mAb (▪), in conjunction with anti-CD28 mAb plus AF (12.5% vol/vol; ■), or in conjunction with anti-CD28 mAb plus purified PP14 · Fcγ1 (48 μg/mL; ▵). [3H]-thymidine incorporation was determined 72 hours later as described in the legend for Figure 1. Comparable results were obtained in 3 experiments. Jacob Rachmilewitz et al. Blood 2001;98:3727-3732 ©2001 by American Society of Hematology

AF increases the dose of peptide antigen required for T-cell activation.PBMCs were stimulated for 24 hours with various MA58-66 peptide antigen concentrations, with or without either AF (10%) or AF immunodepleted of its PP14. AF increases the dose of peptide antigen required for T-cell activation.PBMCs were stimulated for 24 hours with various MA58-66 peptide antigen concentrations, with or without either AF (10%) or AF immunodepleted of its PP14. The number of cells producing IFN-γ was determined by ELISAspot.18 19 Jacob Rachmilewitz et al. Blood 2001;98:3727-3732 ©2001 by American Society of Hematology