Volume 25, Issue 6, Pages 907-918 (December 2006) The Hematopoietic Isoform of Cas-Hef1-Associated Signal Transducer Regulates Chemokine-Induced Inside-Out Signaling and T Cell Trafficking  Adam G. Regelmann, Nichole M. Danzl, Celestine Wanjalla, Konstantina Alexandropoulos  Immunity  Volume 25, Issue 6, Pages 907-918 (December 2006) DOI: 10.1016/j.immuni.2006.09.014 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Chat-H Deficiency Impairs T Cell Migration (A) Immunoblot of Chat-H expression in lysates from T cells transduced with Chat-H-specific shRNAs (CHi2, CHi3) and either left unsorted or GFP-sorted. Expression of Zap-70 was used as a loading control. (B and C) 1–2 × 105 CD4+ T cells transduced with vector (filled symbols) or CHi3 (open symbols) were used in a transwell migration assay as described in the Experimental Procedures. The lower wells contained increasing concentrations of either CXCL12 or CCL21. Percent cells migrated were determined by averaging counted cells from duplicate samples on a hemocytometer. Error bars for (B), (C), and (D) represent standard deviations (SD). One of two independent experiments is shown. (D) CXCL12 (500 ng/ml) was added to the lower chamber only (L), the upper and the lower chamber (U/L), or not added to either chamber (Un), and migration was determined as in (B) and (C). Black and gray bars represent vector- and CHi3-transduced T cells, respectively. (E) Expression of chemokine and integrin receptors in transduced CD4+ T cells was analyzed by staining the cells with specific antibodies, gating on GFP+ cells, and examining receptor expression by flow cytometry. Immunity 2006 25, 907-918DOI: (10.1016/j.immuni.2006.09.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Expression of RNAi-Resistant Chat-H Rescues T Cell Migration (A) T cells were coinfected with viral supernatants carrying a pLL3.7 lentiviral vector expressing YFP instead of GFP and retroviruses expressing HA-tagged rChat-H that contains silent mutations within the region targeted by CHi3 and is resistant to CHi3-mediated downregulation. The percentages of YFP+GFP+ T cells were determined by FACS. (B) Expression of endogenous Chat-H and rChat-H in the presence or absence of CHi3 was analyzed on immunoblots with a Chat-H antibody as in Figure 1. 1–2 × 105 unsorted cells were used for migration assays (bar graph). Percent migration of YFP+GFP+ double-positive cells was determined by counting the total number of cells that migrated in the bottom chamber of triplicate transwells, and comparing GFP+/YFP+ input versus migrated populations by FACS. Bar graphs represent averages ± standard deviation (SD). (C) T cells were infected with vector, CHi3, or the scrambled control shRNA CHi3s and Chat-H expression and migration assays were performed as described in Figures 1A and 1B, respectively. Bar graphs represent averages of triplicate samples ± SD. Immunity 2006 25, 907-918DOI: (10.1016/j.immuni.2006.09.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Defective In Vivo Homing of Chat-H-Depleted Cells (A) T cells were transduced with either vector or CHi3, and surface marker expression on GFP+ cells was analyzed by FACS and compared to freshly isolated CD4+ cells. (B) Transduced T cells were sorted by gating on GFP, and then vector-transduced cells were stained with DDAO and CHi3-transduced cells were stained with CMRA (no difference was observed if the dyes were switched). They were mixed at a 1:1 ratio, and 5 × 106 total cells were injected into the tail veins of syngeneic mice. 1 hr after injection, mice were sacrificed and cells from dissected tissues were analyzed by FACS. Numbers represent percentages of GFP-gated cells (dot plots). (C) Ratios of CHi3:vector for different tissues were compared to the input ratio. When the input ratio deviated from 1.0, input and tissue ratios were multiplied by a correction factor. Bar graphs represent the average of CHi3:vector ratios ± SD for tissues obtained from at least three different mice. BL, blood; LG, lung; ∗p < 0.01; pLN, lymph node; ∗p < 0.05; SP, spleen; ∗p < 0.05; LV, liver. Immunity 2006 25, 907-918DOI: (10.1016/j.immuni.2006.09.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Defective Rap1 Activation and LFA-1-Mediated Adhesion in Chat-H-Depleted Cells (A and B) Cells infected with vector-, CHi3-, or CHi3s-expressing lentiviruses were stimulated with CXCL12 for 30 s, and Rap1- (A) or Rac1-GTP (B) amounts were determined in pull-down assays. Rap1 and Rac1 were visualized on immunoblots with specific antibodies. Efficient Chat-H knockdown was confirmed by immunoblotting with a Chat-H-specific antibody ([B], bottom left), and ZAP-70 expression was used as a loading control ([B], bottom right). Equal amounts of total Rap1 were present in vector- or CHi3-infected T cells as determined by immunoblotting ([A], bottom). WCE, whole cell extract. (C) Stimulated vector (solid line) or CHi3-transduced (dashed line) T cells were plated in triplicate wells, fixed, stained with Alexa Fluor 633-conjugated phalloidin, and analyzed by FACS. Cells were gated on YFP, and MFIs in the 633 channel were collected. MFIs from different time points were divided by the unstimulated MFI to obtain the percent increase in F-actin. Data shown are representative of three separate experiments. Bars represent averages of triplicate wells ± SD. (D) Transduced T cells were left unstimulated or stimulated with CXCL12 for different time points as indicated and plated on ICAM-1-coated plates in the presence or absence of LFA-1 blocking antibody (CD11a, clone M17/4) as shown. Adherent cells were fixed and stained with crystal violet, and the absorbance was determined at 540 nm. Each time point represents the average of at least three experiments ± SD. 5 min, ∗p < 0.05; 10 min, ∗p < 0.01; 20 min, ∗p < 0.01; 30 min, ∗p < 0.005. (E) T cells infected with vector (black bars), CHi3 (gray bars), or CHi3s (white bars) were stimulated with CXCL12 or PMA for 10 min and adhesion assays were performed as in (D). Each bar represents the average of at least three experiments ± SD. Adhesion of CXCL12-induced Chat-H-depleted cells (gray bar) was statistically significant as compared to vector (black bar, ∗p < 0.01) or CHi3s-infected (white bar, ∗p < 0.05) cells. Immunity 2006 25, 907-918DOI: (10.1016/j.immuni.2006.09.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Chat-H Is Required for CXCL12-Induced Y-Phosphorylation and Steady-State S-T Phosphorylation of CasL (A) Transduced CD4+ T cells were either left unstimulated or stimulated with CXCL12 for the indicated times and immediately lysed. Cell lysates were immunoprecipitated with a CasL antibody and blots were probed with the indicated antibodies. Fold induction was determined by quantifying protein bands with Odyssey imaging software (Licor). The bottom two panels show C3G and CasL expression in whole lysates (WCE) of vector- or CHi3-infected T cells. (B) Uninfected expanded T cells were stimulated with CXCL12 as shown, and normalized cell lysates were immunoprecipitated with C3G-specific antibody and blots were probed with indicated antibodies. (C) HEK293 cells were transfected with either vector alone or one that expressed FLAG-tagged Chat-H, Chat, or a Chat-HΔCT mutant, and immunoblots of total lysates were probed for p130Cas and anti-FLAG. (D) Chat-H- and Chat-GFP fusion proteins or GFP vector were expressed in HEK293 cells and analyzed by confocal microscopy (left). Expression of endogenous Chat-H was analyzed in primary T cells infected with CHi3 (GFP) with a Chat-H antibody. The presence of Chat-H is indicated by arrows as an outer ring in an uninfected cell, whereas an infected green cell is shown on the left of the same image. Immunity 2006 25, 907-918DOI: (10.1016/j.immuni.2006.09.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 The N Terminus of Chat-H and Its Interaction with CasL Are Required for T Cell Migration (A) HA-tagged Chat-H and mutants expressed from pMiG vectors were tested for their ability to bind to and phosphorylate p130Cas. HEK293 cells transfected with vector, rChat-H (1), ΔN (2), and Y787E (3) were immunoprecipitated with HA antibody, and immunoblots were probed for p130Cas and HA antibodies (left). The effect of the different Chat-H mutants on CasL phosphorylation was examined in sorted YFP+GFP+ T cells coinfected with vector or CHi3-expressing lentivirus (YFP) and pMiG retroviral vectors (GFP) expressing the different mutants (right). Expression of Zap-70 was used as a loading control. (B) Migration of GFP+YFP+ T cells expressing rChat-H or mutants was examined in vitro with ∼2 × 105 unsorted cells. Percent migration of YFP+GFP+ cells was determined as in Figure 2. Bars represent the average of triplicate samples ± SD. One of three representative experiments is shown. (C) Sorted YFP+/GFP+ T cells were stimulated for 10 min and plated on ICAM-1-coated plates, and percent adhesion was determined as in Figure 4D. Each bar represents the average of at least three experiments. Statistically significant differences in adhesion were observed between Chat-H-reconstituted cells as compared to Chat-H-depleted cells or cells reconstituted with the ΔN or the Y-E mutants. ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < 0.005. Immunity 2006 25, 907-918DOI: (10.1016/j.immuni.2006.09.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 CasL Is Required for T Cell Migration (A) Oligonucleotides homologous to three regions of the CasL cDNA were generated and cloned into a YFP-expressing pLL3.7U vector as described for Chat-H. T cells were transduced with the different constructs and sorted on YFP, and CasL expression was assessed in sorted cells by immunoblots (blots). Migration of vector- or CLi1-3-transduced cells was determined as described in Figure 1. (B and C) T cells were transduced with lentiviral supernatants expressing CLi3 and YFP and retroviruses expressing human CasL (hCasL) or the hCasL-ΔST mutant (B), or CLi1 and GFP or CHi3 and YFP as shown (C) and migration of YFP+GFP+ cells was determined as described in Figure 2. Expression of CasL and Chat-H proteins were analyzed on immunoblots with lysates of sorted YFP+GFP+ cells. Zap70 was included as a loading control. (D) Working model of Chat-H function in T cells. The Chat-H-CasL complex regulates steady-state and chemokine-induced adhesion and migration of T cells. The N-terminal region of Chat-H localizes the complex to the plasma membrane and promotes CasL S-T phosphorylation. Chemokine stimulation triggers CasL tyrosine phosphorylation and the recruitment of a signaling intermediate or complex to the Chat-H-CasL module, which in turn leads to Rap1 activation, integrin-mediated adhesion, and migration. Immunity 2006 25, 907-918DOI: (10.1016/j.immuni.2006.09.014) Copyright © 2006 Elsevier Inc. Terms and Conditions