|
|
Blood, Vol. 114, Issue 3, 580-588, July 16, 2009
CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway
Blood Gollmer et al.
114: 580
Supplemental materials for: Gollmer et al
Materials and methods Reagents
Antibodies (Abs) against mouse CD3ε (145-2C11), CD28 (37.51), CD44 (IM7), CD45.1 (A20), CD62L (MEL-14), CD69 (H1.2F3), IL-2 (JES6-5H4), DO11.10 (KJ1-26), TCRVα2, TCRVβ5, and anti–human-IgG were purchased from BD Pharmingen (San Diego, CA). Human CCL19-Ig was used for CCR7 staining as described.1 All Abs used in western blotting were from Cell signaling technologies (Danvers, MA) unless stated otherwise. Anti-ERK1/2 and phospho-JNK were purchased from Upstate Biotechnology (Lake Placid, NY). Murine CCL21 and CXCL12 as well as human CCL19 and CXCL12 were obtained from Peprotech (London, UK), and murine CCL19 was from R&D Systems (Minneaoplis, MN). Carboxyfluorescein diacetate, succinimidyl ester (CFDA SE; CFSE), 5-chloromethyfluorescein diacetate (CMFDA; Cell Tracker Green) and fura-2 AM were purchased from Molecular probes (Eugene, OR). Pertussis toxin and ionomycin were from Sigma (St. Louis, MO), Wortmannin (WMN), Phorbol-12-myristate-13-acetate (PMA), 3H-Thymidine from GEHealthcare (Glattbrugg, Switzerland). Chicken OVA323–339 (ISQAVHAAHAEINEAGR) and turkey OVA323–339 (ISQAAHAAYAEIYEAGR) were obtained from the National Center for Biotechnology (Madrid, Spain). MCC88–103 (ANERADLIAYLKQATK) was from Genescript (Boston, MA). DBY589–603 peptide (NAGFNSNRANSSRSS) was from NeoMPS (Strasbourg, France). The Pak1 inhibitor IPA-3 was synthetized as described.2 Immunoblotting
Phosphorylation of ERK1/2, JNK, p38, and Akt was assessed by western blotting (1.5–2.5 × 106 cells/lane), using phosphospecific Abs against Thr202/Tyr204 of p44 and p42 ERK, Thr183/Tyr185/Thr221/Tyr223 of p54 and p46 JNK, Thr180/Tyr182 of p38 and Ser473 of Akt and Ser338 of Raf, followed by ECL detection (Cell Signaling, Danvers, MA). For detection of Ras-GTP and Rac-GTP, immunoprecipitations with the Raf-RBD and PAK21-RBD were carried out as described.3 In some experiments, western blots were developed using dye-conjugated secondary Abs (Odysse IRDye 800 or Odysse IRDye 680 goat anti-rabbit or anti-mouse Ab) with the LI-COR Odyssey Infrared Imaging System according to the manufacturer's instruction (LI-COR Biosciences, Bad Homburg, Germany). PLN slice preparation
PLN slice preparation was performed as previously described4 with minor modifications. In brief, agarose-embedded PLN from WT mice were cut into 320 µm-thick slices with a vibratome (VT1200S, Leica) and transferred to 0.4 µm organotypic culture inserts (Millicell, Millipore). CD4+ T cells were isolated from the peripheral and mesenteric LN of Marilyn TCR tg mice and incubated for 3 min at 37°C with 0.25 µM 5-chloromethylfluorescein diacetate (CMFDA) or 45 min at 37°C with 2 µM fura 2-AM (both from Molecular Probes) in HBSS. Cells were then washed in RPMI/10% FCS and resuspended in this medium. In some experiments, T cells were pretreated with 100 ng/ml PTX or the B subunit of the toxin (suB; Calbiochem) for 10 min at 37°C and washed twice in RPMI/10% FCS. A total of 2 × 105 lymphocytes in 10–20 µl were added onto the cut surface of each slice. To concentrate the cells on the tissue, a stainless steel ring was placed to the agarose surrounding the slice. Slices were then incubated for 1 h at 37°C, 6% CO2, gently washed to remove residual cells that have not entered tissue and kept at 37°C, 6% CO2 before imaging. Isolation and activation of human T cells
Human PBMCs were obtained from whole blood of healthy human donors in agreement with “Etablissement Français du Sang” guidelines. Peripheral blood T cells were purified from PBMCs by negative selection using a T-cell isolation kit (BD Biosciences). In some experiments, T cells were treated with 100 nM Wortmannin for 30 min at 37°C. T cells were stimulated with CCL19 and anti-CD3 for the indicated times, then processed for FACS analysis.
Files in this Data Supplement:
- Table S1. Quantification of western blot analysis in primary T cells (PDF, 27.4 KB) -
The signal average of the “0” time point was arbitrarily put to “1,” and the signal intensities at various time points, and treatments was adjusted accordingly. Signals in costimulated samples (CCL21 + TCR) representing more than double the sum of single-stimulated (CCL21 or TCR) are shown in bold. All values are mean ± SEM. The occasionally high SEM was due to variations in absolute fold induction between individual experiments, while the patterns of signal molecule activation as shown in Figs. 3 and 5 remained consistent between experiments. To normalize for this variability, we also calculated the “fold increase” of costimulated over single-stimulated cells as shown in the lower panel of the table for Rac- and Ras-GTP formation and ERK phosphorylation.
- Figure S1 (JPG, 237 KB)
-
(A) Soluble CCL21 does not increases the migration of T cells in contact with splenocytes. CMFDA-loaded T cells were cocultured with splenocytes and imaged during 10 min. Cells were treated or not with 1 µg/ml CCL21 added 5 min before imaging. T-cell displacements measured in PLN slices are also presented for comparison. The red line indicates average velocity. (B) CCL21-induced elongation of CMFDA-labeled T cells. The left panel shows a representative micrograph of T cells prior to adding CCL21 (top) and 5 min after CCL21 addition (bottom). The right panel shows the shape index calculated using the Morphometric Analysis Function of Metamorph.
- Figure S2 (JPG, 70.4 KB)
-
(A) CCL19 mediates PI3K-independent costimulation of human T cells. Fura-2 loaded human T cells preincubated with 100 nM WMN for 30 min or medium alone were stimulated at the arrow with 5 µg/ml anti-CD3 in the presence or absence of 0.5 µg/ml CCL19. Ca2+ signal was measured on cell population with a spectrofluorimeter. (B) Percentage of CD69+ human T cells stimulated with 5 µg/ml anti-CD3 in the presence or absence of 0.5 µg/ml CCL19 or CCL21. CD69 expression was measured by flow cytometry 12 h later. The red number indicates fold increase in presence of chemokine. Data are representative of two to three experiments.
- Figure S3 (JPG, 195 KB)
-
(A) Expression of CD69 and CD25 in absence of DOCK2. 2B4 control or DOCK2-deficient 2B4 cells were stimulated using irradiated splenocytes loaded with indicated concentrations of MCC peptide, in presence (black bars) or absence (white bars) of 100 nM CCL21. CD69 and CD25 surface expression was measured 8 and 16 h later, respectively. (B) Normal Akt phosphorylation in absence of DOCK2. 2B4 control and DOCK2-deficient T cells were stimulated and analyzed for phosphorylation of Akt as in Figs. 3 and 5. One representative blot of five is shown.
- Figure S4. Reduced CCL21-mediated costimulation in Pak1-inhibited CD4+ T cells (JPG, 84.2 KB)
-
OT-II TCR tg CD4+ T cells were stimulated using irradiated splenocytes loaded with indicated amounts of OVA peptide, in presence of increasing amounts of the Pak1 inhibitor IPA-3. After 72 h, proliferation was measured using 3H thymidine incorporation. The numbers indicate the fold increase over control (“ctrl” = no chemokine). Shown are quadruple values from one of three similar experiments.
- Figure S5. Antigen-induced T-cell responses measured in PLN slices (JPG, 94.5 KB)
-
(A) Ca2+ responses (black) and motility (red) in one representative Marylin TCR-tg CD4+ T cell within a PLN slice. T cells were loaded with fura-2 and added to a PLN slice 30 min before the recording. After several minutes of imaging, the preparation was perfused with a solution containing 1 µM DBY peptide. The dotted line represents the curve-fitted velocity. A time-lapse animation of Ca2+ responses is shown in Video 3. (B) Percentage of CD69+ cells as a function of the DBY peptide concentration. CMFDA-labeled Marylin TCR-tg T cells were overlaid on PLN slices. Two h after DBY peptide stimulation, CD69 expression was determined on CMFDA+ CD45.1+ cells. (C) Kinetics of CD69 expression in PLN slices treated with 100 nM DBY peptide. Results are representative of three experiments.
- Video 1. Motility of CMFDA-labeled CD4+ T cells on irradiated splenocytes in absence of CCL21 (AVI, 3.18 MB)
-
The movie is an overlay of the unlabeled splenocytes and the fluorescently labeled CD4+ T cells.
- Video 2. Motility of CMFDA-labeled CD4+ T cells on irradiated splenocytes in presence of CCL21 (AVI, 3.16 MB)
-
The movie is an overlay of the unlabeled splenocytes and the fluorescently labeled CD4+ T cells.
- Video 3. Motility and Ca2+-flux of Marylin TCR tg CD4+ T cells after DBY peptide superfusion of PLN slices (AVI, 3.39 MB)
-
Fura-2–loaded Marylin TCR tg CD4+ T cells inside PLN slices were tracked before and after addition of 1 µM DBY peptide to the PLN slice (start of superfusion indicated by “HY peptide”). Ca2+-flux induced by peptide superfusion inversely correlates with cell motility.
REFERENCES
1. Stein JV, Soriano SF, M'Rini C, et al. CCR7-mediated physiological lymphocyte homing involves activation of a tyrosine kinase pathway. Blood. 2003;101:38–44.
2. Deacon SW, Beeser A, Fukui JA, et al. An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chem Biol. 2008;15:322–331.
3. Nombela-Arrieta C, Lacalle RA, Montoya MC, et al. Differential Requirements for DOCK2 and Phosphoinositide-3-Kinase gamma during T and B Lymphocyte Homing. Immunity. 2004;21:429–441.
4. Asperti-Boursin F, Real E, Bismuth G, Trautmann A, Donnadieu E. CCR7 ligands control basal T-cell motility within lymph node slices in a phosphoinositide 3-kinase–independent manner. J Exp Med. 2007;204:1167–1179.
|
|
