Blockade of CD11a by Efalizumab in Psoriasis Patients Induces a Unique State of T-Cell Hyporesponsiveness  Emma Guttman-Yassky, Yulia Vugmeyster, Michelle A. Lowes, Francesca Chamian, Toyoko Kikuchi, Mark Kagen, Patricia Gilleaudeau, Edmund Lee, Brisdell Hunte, Kathy Howell, Wolfgang Dummer, Sarah C. Bodary, James G. Krueger  Journal of Investigative Dermatology  Volume 128, Issue 5, Pages 1182-1191 (May 2008) DOI: 10.1038/jid.2008.4 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Inhibition of ex vivo anti-CD3-, anti-CD2, and anti-CD3/anti-CD28-induced activation of circulating T cells in efalizumab-treated psoriatic patients. (a) CD69 levels (MFI) on T cells stimulated ex vivo with increasing concentrations of anti-CD3 antibody at different time points during (up to day 84) and after efalizumab treatment. (b) CD11a expression on unstimulated T cells to assess CD11a saturation throughout the treatment period (up to day 84) and upon efalizumab washout. (c) A set of representative dot plots illustrates that efalizumab treatment by week 2 inhibits T-cell activation (CD69 MFI) induced by all three cell-surface stimuli, although less so with anti-CD3/CD28 stimuli. (d) Mean percent change in CD69 MFI with various stimuli, in patients on efalizumab treatment compared with baseline. *Indicates P<0.05. (e) Mean change in CD69 MFI with PMA/ionomycin stimulation on days 0 and 14 of efalizumab treatment showing no difference in T-cell activation by this stimuli. (f) A representative CD69 histogram for PMA/ionomycin stimulation illustrates that efalizumab treatment (black line, day 14) has no effect on PMA/ionomycin-mediated upregulation of CD69, as compared with baseline (gray line, day 0). Dotted line corresponds to the baseline CD69 expression on unstimulated T cells. Journal of Investigative Dermatology 2008 128, 1182-1191DOI: (10.1038/jid.2008.4) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Inhibition of ex vivo anti-CD3- and anti-CD2-induced IFN-γ production by T cells in efalizumab-treated psoriatic patients. (a) T cells’ ability to synthesize IFN-γ in response to anti-CD3 antibody after 2 weeks of efalizumab treatment is highly diminished (P<0.02). (b) Representative FACS data for three different T-cell activation stimuli, anti-CD3, anti-CD2, PMA/ionomycin, at baseline and week 2 treatment with efalizumab. Efalizumab affected T-cell ability to produce IFN-γ in response to CD3 ligation but not to PMA/ionomycin stimulation. (c) Percent change in IFN-γ+ T cells in all study patients. Black lines correspond to individual patients. Journal of Investigative Dermatology 2008 128, 1182-1191DOI: (10.1038/jid.2008.4) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Assessment of intracellular signalling through TCR/CD3 complex. Normal anti-CD3-mediated intracellular Ca2+ flux response in T cells from efalizumab-treated patient PBMCs that were loaded with Indo-1, activated with anti-CD3, and cross-linked with an anti-mouse IgG. The Ca+2 flux was measured as the ratio between bound and unbound Indo-1 versus time. (a) Intracellular Ca+2 flux from a representative normal volunteer pre- and post-stimulation with OKT3 (anti-CD3) and T11 (anti-CD2). (b and c) T cell intracellular Ca+2 flux responses from two representative patients are presented before and after efalizumab therapy. Journal of Investigative Dermatology 2008 128, 1182-1191DOI: (10.1038/jid.2008.4) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Efalizumab-mediated inhibition of in vitro anti-CD3- and anti-CD3/anti-CD28-induced T-cell activation and the reversal of this inhibition by exogenous IL-2. (a) Anti-CD3-induced T-cell activation in the presence of the indicated concentration of efalizumab was measured by monitoring CD69 expression (geometric MFI) on CD2+ lymphocytes in whole blood obtained from normal healthy volunteers. A bar indicates efalizumab in vivo levels in psoriatic patients during therapeutic administration. (b) Anti-CD3-dependent PBMC proliferation in the presence of control IgG, efalizumab, monoclonal antibody MHM24 (murine parent of efalizumab), or Fab fragment of efalizumab was measured. (c) Anti-CD3/anti-CD28-dependent T-cell proliferation in the presence of efalizumab (1μgml−1) with and without IL-2 was assessed. Panels (b) and (c) show mean data (n=3), with error bars showing standard deviations. Journal of Investigative Dermatology 2008 128, 1182-1191DOI: (10.1038/jid.2008.4) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Down-modulation of multiple cell-surface molecules in T cells from peripheral blood and lesional skin in efalizumab-treated psoriatic patients. (a) Mean level (MFI) of surface molecules on T cells at baseline and after 2 weeks of treatment for peripheral blood (study 1, open square; study 2, gray square) and lesional skin (black square). Asterisks (*) indicate that relative MFI values were statistically different compared with baseline, *P<0.05, **P<0.01, ***P<0.001. (b–g) A representative set of FACS dot plots from an individual patient (gated on T cells, except for panels (d) and (e)) is shown for peripheral blood (left) and lesional skin (right) at baseline and day 14. Quadrants were set on the basis of appropriate isotype controls. A fraction of positive cells in the indicated quadrants or an MFI value is shown. In panel (g), MFI values were calculated for T cells in the circled gate (CD29hi). Journal of Investigative Dermatology 2008 128, 1182-1191DOI: (10.1038/jid.2008.4) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Down-modulation of VLA-4 and CD8 in peripheral blood CD8+ T cells in efalizumab-treated psoriatic patients. (a) Histograms for a representative patient illustrate bimodal distribution of VLA-4 (CD49d/CD29) at baseline (gray line) on CD8+ cells, and a subsequent shift in VLA-4hi/VLA-4lo ratio after 2 weeks of treatment. (b) Individual patient data were used to calculate the mean CD49d MFI for each time point and for each CD3+CD8+ subset, as indicated. Dotted line corresponds to baseline CD49d MFI value for the CD49dlo subset. (c) CD8 levels on all CD3+CD8+ cells were quantified throughout the treatment period (12 weeks) and upon efalizumab washout. Journal of Investigative Dermatology 2008 128, 1182-1191DOI: (10.1038/jid.2008.4) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions