Hidenobu Kanda, Rebecca Newton, Russell Klein, Yuka Morita, Michael D. Gunn & Steven D. Rosen

Transcription

1 Autotaxin, a lysophosphatidic acid-producing ectoenzyme, promotes lymphocyte entry into secondary lymphoid organs Hidenou Kanda, Reecca Newton, Russell Klein, Yuka Morita, Michael D. Gunn & Steven D. Rosen a peptide 1.5 Rait IgG IgG (µg/ml) + Peptide +Peptide Rait IgG IgG Rait Supplementary figure 1. Characterization of peptide antiody against ATX. (a) Reactivity of the affinity-purified peptide antiody to ratx protein was determined y ELISA. ratx protein (1 ng/well) was coated on an Immulon 2HB plate and the wells were incuated with the indicated concentrations of normal rait IgG, anti-atx pa or anti-atx pa preincuated with the immunogen peptide (2 µg/ml). Color was developed after incuating with iotin-conjugated goat anti-rait IgG and streptavidin-alkaline phosphatase. () Adjacent cryostat sections of mouse MLN were reacted with anti-atx pa (left), anti-atx pa preincuated with the immunogen peptide (1 µg/ml) (middle) or isotype rait IgG control (right). The antiodies were at a concentration of 5 µg/ml. Color was developed with a iotin conjugated goat anti-rait IgG and Cy2-streptavidin. The experiment in a was performed twice. The results in are representative of three independent experiments.

2 ATX MECA-79 Merged BF NOD RIP-BLC Supplementary figure 2. Localization of ATX protein in HEV-like vessels within tertiary lymphoid organs. Two-color immunofluorescence was performed on cryostat sections of pancreata of an NOD mouse (1 weeks, female) and RIP-BLC mouse (4 weeks, female) for ATX (green) and MECA-79 reactivity (red). Bright field images of the same fields (hematoxylin staining) are presented on the right. Pancreata from 4 NOD mice (one diaetic and three non-diaetic) gave equivalent results. A single RIP-BLC mouse was analyzed. The ar denotes 1 µm.

3 a Mn 2+ LDV RGD LDV+RGD α4 αl α4+ αl Adhesion to VCAM-1 (% of total input) LDV RGD LDV+RGD Mn 2+ α4 αl α4+ αl c LDV Adhesion to ATX (% of total input) RGD LDV+RGD EDTA α4 αl α4+ αl Adhesion to ATX (% of total input) Supplementary figure 3. Binding of mouse lymphocytes to ATX and VCAM-1. Thymocytes were preincuated with the indicated function-locking As (1 µg/ml), with the indicated integrin-inding peptides (1 µm) or with no additives ( none ). Adhesion of thymocytes to svcam-1 (murine, 1 ng) and ATX (human, 1 ng) in the presence of Mn +2 is shown in a and, respectively. With no adhesive sustrate coated, the numer of adhesive cells was 5% of the input cells. The α 4 antiody inhiited adhesion of cells to VCAM-1 ut none of the antiodies affected adhesion to ATX. The LDV peptide had a partial affect on adhesion to VCAM-1 ut not to ATX. In c, adhesion of thymocytes to ATX in the presence of 1 mm EDTA is shown. (a c) Data are presented as means ± SD s from quadruplicate wells. Similar results were otained for mouse splenocytes. The amino acid sequences of mature human and murine ATX are 95% identical. Both proteins have RGD and LDV sequences in the same relative positions. The results shown are representative of 4 independent experiments with either splenocytes or thymocytes.

4 a 3 25 Migrated cells (% of input) LPA (nm) Migrated cells (% of input) LPA+PTX LPA c 12 1 Migrated cells (% of input) LPA, upper: CXCL12, lower: ng/ml 1 ng/ml Supplementary figure 4. LPA-induced migration of mouse lymphocytes. (a) LPA was added to the upper chamer of transwell units and tested for its effects on PLN lymphocyte migration to the lower chamer. () The effect of PTX pretreatment (2 ng/ml, 2 h) on the lymphocyte response to LPA (1 µm) was determined. (c) The responses of lymphocytes to the indicated treatments with LPA (1 µm) and CXCL12 (5 or 1 ng/ml) were evaluated in the transwell assay. * denotes that P <.1 for the indicated comparison. The results of a and are representative of 3 and 2 independent experiments, respectively. The experiment in c was performed once.

5 Supplementary figure 5. Model of ATX function in lymphocyte homing. ATX is secreted from the apical surface of HECs (Step 1) and then inds to activated α4β1 (denoted y asterisk) on arrested human T-cells (and to receptors of unknown identity on mouse T- cells) (Step 2). ATX may also e immoilized on the surface of HEVs, which could enhance its interaction with lymphocytes through avidity effects. Through its lysopld activity, the lymphocyte-targeted ATX acts on LPC in the plasma to produce a high concentration of LPA in the vicinity of the lymphocyte (Step 3). The LPA triggers intracellular signaling in the lymphocyte and promotes its migration into the lymph node (Step 4). The locally-produced LPA could also potentially exert effects on the endothelial cells to enhance the recruitment process. We propose that enzymatically inactive ATX (T21A) exerts a dominant negative effect y competing with endogenous ATX for a limited numer of receptor sites on the lymphocyte (and possily on the HEC), thus limiting the activation of LPA signaling.

6 a Protein stain (kda) LysoPLD activity WT T21A Buffer ATX (µg/ml) c WT T21A svcam Sustrate (ng/well) Supplementary figure 6. Production and characterization of recominant ATX proteins. (a) WT ATX and T21A ATX were purified from transfected COS-7 cells and analyzed y SDS-PAGE for purity (protein stain) and for reactivity with the ATX antiody. () The LysoPLD activities of WT ATX and T21A ATX were determined at the indicated protein concentrations. The values shown are means ± SD s ased on triplicate determinations. (c). WT ATX, T21A ATX, and VCAM-1 were tested for their ailities to support adhesion of mouse T cells in the presence of Mn 2+. Error ars were omitted in c as SD s were less than 5 % of the means. Scale ars, 1 µm. The results in a are representative of 4 independent experiments and in of 2 independent experiments.