Adhesion and migration of T cells are controlled by chemokines and by adhesion molecules, especially integrins, and have critical roles in the normal physiological function of T lymphocytes. Using an RNA-mediated interference screen, we identified the WNK1 kinase as a regulator of both integrin-mediated adhesion and T cell migration. We found that WNK1 is a negative regulator of integrin-mediated adhesion, whereas it acts as a positive regulator of migration via the kinases OXSR1 and STK39 and the ion co-transporter SLC12A2. WNK1-deficient T cells home less efficiently to lymphoid organs and migrate more slowly through them. Our results reveal that a pathway previously known only to regulate salt homeostasis in the kidney functions to balance T cell adhesion and migration.
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We thank S. Ley for critical reading of the manuscript; D. Alessi (University of Dundee) for Oxsr1T185A mice, anti-pSer325-OXSR1 antibodies and discussions; R. Zamoyska (University of Edinburgh) for Jurkat cells, D. Bell (Francis Crick Institute) for help with image analysis; M. Abadier (Theodor Kocher Institute) for isolation and cultivation of primary mouse brain microvascular endothelial cells; N. Hogg (Francis Crick Institute) for Kim127 and m24 antibodies; and L. Satlin and C. Else (Mount Sinai Medical Center) for access to Slc12a2 mutant mice (supported by the NIH NIDDK grant P30 DK079307, Pittsburgh Center For Kidney Research, Core B). Supported by the Francis Crick Institute (V.T.), which receives its core funding from the Medical Research Council, Cancer Research UK and the Wellcome Trust, the Medical Research Council (U117527252 to V.T.), the Wellcome Trust (089185 to R.K. and V.T.), the Biotechnology and Biological Sciences Research Council (BB/L00805X/1 to R.K. and V.T.), the US National Institute of Health (DK59530 to C.-L.H.) and the Swiss Multiple Sclerosis Society (R.L.).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 The WNK1–OXSR1-STK39–SLC12A2 pathway regulates adhesion and migration of Jurkat T cells.
Expression of the WNK1, OXSR1, STK39 and SLC12A2 genes was knocked down in Jurkat T cells by transfection of either pools of siRNAs or individual siRNAs. a, Mean (±SEM) levels of mRNA for indicated genes remaining 72 h after transfection with either pools (n≥4) or individual (n≥3) siRNAs against the indicated genes. The purple columns indicate simultaneous knock down of both OXSR1 and STK39 either with pools of siRNAs, or individual siRNAs. (b, c) WNK1 expression was knocked down in Jurkat cells with 2 individual siRNAs. Mean±SEM conjugation of Jurkat T cells to SEE-pulsed NALM6 B cells (n=4) normalized to the maximum response of cells transfected with NT siRNAs (b) and mean±SEM binding of soluble ICAM1 complexes following stimulation of Jurkat T cells with CXCL12, anti-CD3 or MnCl2 or unstimulated (US) (c, n=4, control; n=6, mutant). (d, e) Mean±SEM adhesion to plate-bound ICAM1 of Jurkat T cells transfected with non-targeting (NT) siRNAs, pools of siRNAs against WNK1 or LCK (n=6), or individual siRNAs against WNK1 (n=5) (d), or siRNA pools against WNK1, OXSR1 and STK39 together and SLC12A2 (n=4) (e). Graphs were normalized to the maximum response of cells transfected with NT siRNAs. (f), Mean±SEM surface levels of CXCR4, TCR and LFA-1 on Jurkat T cells in which expression of the indicated genes had been knocked down using pools of siRNAs, as indicated by geometric mean fluorescence intensity (GMFI) derived from flow cytometric analysis (n=3-6). (g) CXCL12 and TCR-induced activation of RAP1 in Jurkat T cells in which knockdown of WNK1 was induced by transfection of individual siRNAs. RAP1 activation was measured by pull-down of RAP1-GTP and immunoblotting with anti-RAP1 antibody as shown. At the same time total cytoplasmic lysates were immunoblotted for levels of total RAP1. Graph shows mean±SEM amount of RAP1-GTP in the pull-down normalized to total levels of RAP1 in the cell (n=3, CCL21 stimulation; n=3, anti-CD3 stimulation). (h) Mean±SEM migration of Jurkat T cells through Transwells in response to CXCL12; expression of the indicated genes was knocked down using individual siRNAs (n=4). Signals were normalized to CXCL12-induced migration of cells transfected with NT siRNAs.
Supplementary Figure 2 Expression of surface proteins on T cells deficient in WNK1 or SLC12A2.
a-c, Mean±SEM percentage of Wnk1 mRNA remaining in CD4+ or CD8+ splenic T cells from control or conditional WNK1-deficient mice: Wnk1+/+dLck-Cre and Wnk1fl/+dLck-Cre mice (n=8, control; n=7, mutant) (a), radiation chimeric mice generated by reconstituting RAG1-deficient mice with bone marrow from Wnk1fl/+RCE or Wnk1fl/flRCE mice (n=5) (b) or with bone marrow from Wnk1fl/+RCE or Wnk1fl/-RCE mice (n=6) (c) and then treated with tamoxifen (Tamo) or not and analyzed 7 d after start of tamoxifen treatment. d-e, Surface expression of CD62L, CCR7, TCR, and LFA-1 on CD4+CD44-CD62L+ splenic T cells from the radiation chimeras described in c, gated as shown in 2D plots. Expression was analyzed by flow cytometry (d) and quantitated showing mean±SEM geometric mean fluorescence intensity (GMFI) (n=6) (e). f-g, Surface expression of CD62L, CCR7, TCR, and LFA-1 on CD4+CD44-CD62L+ splenic T cells from radiation chimeras generated by reconstituting RAG1-deficient mice with bone marrow from SLC12A2-deficient mice gated as shown in 2D plots. Expression was analyzed by flow cytometry (f) and quantitated showing mean±SEM geometric mean fluorescence intensity (GMFI) (n=5) (g).
Supplementary Figure 3 WNK1 is not required for calcium flux, ERK activation and actin polymerization.
(a, b) Jurkat T cells transfected with non-targeting (NT) siRNA, or siRNA pools against WNK1 or LCK were analyzed for anti-CD3-induced Ca2+ flux (1 of 4 experiments) (a), and for CXCL12 or anti-CD3-induced phosphorylation of ERK1 and ERK2 (n=4, CXCL21 stimulation; n=3 anti-CD3 stimulation) (b). Graphs show mean±SEM of quantitated immunoblot signals for pERK1/2 normalized to ERK1 and ERK2. (c) Mean±SEM levels of F-actin in WNK1-deficient and control CD4+ mouse T cells stimulated with CCL21 (n=4).
Supplementary Figure 4 WNK1-deficient T cells have larger turning angles during interstitial migration in lymph node parenchyma.
Frequency distribution of turning angles during interstitial migration of control or WNK1-deficient T cells in lymph node parenchyma, determined by intravital microscopy following transfer of the T cells into mice in which LFA-1 and VLA-4 integrins were blocked (or not) subsequent to transfer (n=4). Numbers show mean±SEM turning angles.
Supplementary Figure 5 Generation of mice with Wnk1D368A allele.
(a) Diagram shows the wild-type (WT) allele of Wnk1, indicating exons 1 to 4 (E1 – E4) of 28 exons. The 5’ untranslated region of exon 1 is indicated in white, coding regions of exons are in black. The targeting vector contained two DNA fragments homologous to the Wnk1 gene, containing either exon 3 or exon 4, separated by a neomycin resistance gene (Neo) flanked by Frt sites. The sequence of exon 3 in the targeting vector was mutated to introduce the D368A mutation. Beyond the second homology region, a diphtheria toxin A gene (DTA) was added in order to select against non-homologous targeting. Homologous recombination of the targeting vector into the mouse genome resulted in the Wnk1D368A-neo allele, which contained both the D368A mutation in exon 3 and the Frt-flanked Neo gene in intron 3. Mice bearing the Wnk1D368A-neo allele were crossed to a transgenic mouse expressing Flp recombinase in the germline to delete the Frt-flanked Neo gene, resulting in the Wnk1D368A allele as shown, which has the D368A mutation in exon 3 and an Frt site in intron 3. (b) Expected and actual numbers of wild-type Wnk1+/+ (WT), heterozygous Wnk1D368A/+ (Het) and homozygous Wnk1D368A/D368A (Hom) mice identified at weaning, resulting from an intercross of heterozygous mice. The actual yield of homozygous mutant mice was significantly different from the expected yield (Fisher’s exact test). (c) Levels of Wnk1 mRNA assayed using qPCR for mRNA spanning from exon 1 to exon 2 or from exon 5 to exon 6. RNA was purified from splenic T cells taken from mice of the indicated genotypes. Each point represents the result from a single mouse. AU, arbitrary units.
Supplementary Text and Figures
Supplementary Figures 1–5 (PDF 1219 kb)
WNK1-deficient T cells have a reduced speed of CCL21- induced chemokinesis
CD44lowCD4+ T cells from radiation chimeras reconstituted with bone marrow from Wnk1fl/+RCE and Wnk1fl/- RCE mice were labeled with CMFDA and imaged every 10 s for 20 min in the presence or absence of 200 ng/ml CCL21 on ICAM1-coated dishes. (MOV 10889 kb)
3D lymph node histology
C57BL/6J mice were injected with control (WNK1+/-) and WNK1- deficient (WNK1-/-) T cells isolated from radiation chimeras reconstituted with bone marrow from Wnk1fl/+ RCE and Wnk1fl/- RCE mice, and fluorescently labeled with CMAC (blue) and CMTMR (red) respectively. After 20 min recipient mice were injected with an anti-CD62L antibody (MEL14) to block adhesion to high endothelial venules (HEVs) and hence further transmigration. Popliteal lymph nodes were isolated and fixed a further 20 min later. HEVs were revealed with anti-PNAd antibody (grey). Fixed lymph nodes were imaged by multi-photon microscopy and movie shows 3D volume rendering of the resulting data (MOV 14645 kb)
Reduced migration speed and diapedesis of WNK1-deficient T cells on an endothelial cell monolayer under flow
CD44low T cells from radiation chimeras reconstituted with bone marrow from Wnk1fl/+ RCE and Wnk1fl/- RCE mice were labeled with CFSE (WNK1+/-, blue) and CMTMR (WNK1-/-, red), respectively. Cells were allowed to adhere on the endothelial cell monolayer for 4 min at 0.1 dyn/cm2 shear stress, followed by shear at 1.5 dyn/cm2 for another 12 min. Images were taken every 10 s (MOV 3376 kb)
WNK1-deficient T cells have a reduced speed of interstitial migration in the parenchyma of C57BL/6J lymph nodes.
CD44low T cells from radiation chimeras reconstituted with bone marrow from Wnk1fl/+RCE and Wnk1fl/-RCE mice were labeled with CMAC (WNK1+/-, blue) and CMTMR (WNK1-/-, red), respectively, and injected into C57BL/6J recipient mice. HEV was labeled by injection of an Alexa633-conjugated anti-PNAd antibody (grey). Next day migration was imaged in popliteal lymph nodes. Images were taken every 20 s for 30 min (MOV 9311 kb)
SLC12A2-deficient T cells have a reduced speed of CCL21-induced chemokinesis
CD44lowCD4+ T cells from radiation chimeras reconstituted with bone marrow from Slc12a2+/+ and Slc12a2-/- mice were labeled with CMFDA and imaged every 10 s for 20 min in the presence or absence of 200 ng/ml CCL21 on ICAM1-coated dishes. (MOV 10198 kb)
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Köchl, R., Thelen, F., Vanes, L. et al. WNK1 kinase balances T cell adhesion versus migration in vivo. Nat Immunol 17, 1075–1083 (2016). https://doi.org/10.1038/ni.3495