CRK proteins selectively regulate T cell migration into inflamed tissues
Yanping Huang, … , Taku Kambayashi, Janis K. Burkhardt
Yanping Huang, … , Taku Kambayashi, Janis K. Burkhardt
Published January 26, 2015
Citation Information: J Clin Invest. 2015;125(3):1019-1032. https://doi.org/10.1172/JCI77278.
View: Text | PDF
Research Article Immunology

CRK proteins selectively regulate T cell migration into inflamed tissues

Abstract

Effector T cell migration into inflamed sites greatly exacerbates tissue destruction and disease severity in inflammatory diseases, including graft-versus-host disease (GVHD). T cell migration into such sites depends heavily on regulated adhesion and migration, but the signaling pathways that coordinate these functions downstream of chemokine receptors are largely unknown. Using conditional knockout mice, we found that T cells lacking the adaptor proteins CRK and CRK-like (CRKL) exhibit reduced integrin-dependent adhesion, chemotaxis, and diapedesis. Moreover, these two closely related proteins exhibited substantial functional redundancy, as ectopic expression of either protein rescued defects in T cells lacking both CRK and CRKL. We determined that CRK proteins coordinate with the RAP guanine nucleotide exchange factor C3G and the adhesion docking molecule CASL to activate the integrin regulatory GTPase RAP1. CRK proteins were required for effector T cell trafficking into sites of inflammation, but not for migration to lymphoid organs. In a murine bone marrow transplantation model, the differential migration of CRK/CRKL-deficient T cells resulted in efficient graft-versus-leukemia responses with minimal GVHD. Together, the results from our studies show that CRK family proteins selectively regulate T cell adhesion and migration at effector sites and suggest that these proteins have potential as therapeutic targets for preventing GVHD.

Authors

Yanping Huang, Fiona Clarke, Mobin Karimi, Nathan H. Roy, Edward K. Williamson, Mariko Okumura, Kazuhiro Mochizuki, Emily J.H. Chen, Tae-Ju Park, Gudrun F. Debes, Yi Zhang, Tom Curran, Taku Kambayashi, Janis K. Burkhardt

×

Figure 2

CRK/CRKL-deficient T cells show impaired integrin-dependent adhesion.

Options: View larger image (or click on image) Download as PowerPoint
CRK/CRKL-deficient T cells show impaired integrin-dependent adhesion. Ni...
Ninety-six-well plates were coated with 1 μg/ml recombinant mouse ICAM-1 (A) or 3 μg/ml fibronectin (FN) (B). Preactivated WT and CRK/CRKL Dko CD4+ T cells were stained with Calcein-AM, applied to ICAM-1– or fibronectin-coated plates, and allowed to warm to 37°C for 10 minutes without stimulus (No) or in the presence of 10 nM CXCL12, 10 nM CCL21, 1 μg/ml anti-CD3, or 10 ng/ml PMA. Unbound cells were washed off, and adherent cells were quantified using a fluorescence microplate reader. Data represent averages ± SD of triplicate samples from 1 experiment, representative of 5 separate experiments. *P < 0.05, **P < 0.01. (C) Preactivated WT and CRK/CRKL Dko CD4+ T cells were stained with anti-CD11a–FITC or anti-CD29–PE and analyzed by flow cytometry. Representative data from 3 experiments are shown. (D and E) Preactivated WT and CRK/CRKL Dko CD4+ T cells were applied to fibronectin-coated coverslips. After incubation at 37°C for 30 minutes, cells were fixed and analyzed by differential interference contrast (DIC) microscopy. Arrows indicate polarized T cells with a clear uropod. (E) Cells prepared as in D were quantified. Data are average ± SD values from 3 experiments, totaling 433 cells for WT and 387 for CRK/CRKL Dko. *P < 0.05. Scale bar: 20 μm. Statistical analysis was performed using paired (A and B) or unpaired (E) 2-tailed Student’s t tests.