T cell migration requires ion and water influx to regulate actin polymerization

Domain: earth_science
Published: 2023-12-06
Editorial outcome: major_revision

Abstract
Migration of T cells is essential for their ability to mount immune responses. Chemokine-induced T cell migration requires WNK1, a kinase that regulates ion influx into the cell. However, it is not known why ion entry is necessary for T cell movement. Here we show that signaling from the chemokine receptor CCR7 leads to activation of WNK1 and its downstream pathway at the leading edge of migrating CD4+ T cells, resulting in ion influx and water entry by osmosis. We propose that WNK1-induced water entry is required to swell the membrane at the leading edge, generating space into which actin filaments can polymerize, thereby facilitating forward movement of the cell. Given the broad expression of WNK1 pathway proteins, our study suggests that ion and water influx are likely to be essential for migration in many cell types, including leukocytes and metastatic tumor cells.

Introduction
Migration is an essential aspect of T cell physiology. Naive T cells migrate between lymphoid organs in search of antigen, a process that is essential for adaptive immune responses1. T cells exit the bloodstream and enter lymph nodes in response to the chemokine CCL21 presented on blood vessel endothelium. CCL21, acting through the CCR7 receptor on T cells, triggers integrin-mediated adhesion between the T cell and the endothelium. T cells then transmigrate through the endothelium and enter the parenchyma of the lymph node. Here they continue to migrate rapidly in response to CCL19 and CCL21 signaling through CCR7, now predominantly using an integrin-independent mode of migration to scan antigen-presenting cells for the presence of cognate antigen, a process that is critical for an efficient immune response2.

Chemokine-induced migration of T cells is characterized by polarization of the cell, with the formation of a pseudopodium at the leading edge of the migrating cell. This structure contains a dense and highly dynamic network of branched actin filaments which is essential for migration3. Chemokine receptor signaling at the leading edge through phosphatidylinositol 3-kinase (PI3K) results in activation of RAC1, RAC2 and CDC42 GTPases which promote actin polymerization and the branching of actin filaments. Actin polymerization pushing at the leading edge in turn provides the motor that drives the plasma membrane forward, resulting in migration.

WNK1 is a member of the WNK-family of serine/threonine protein kinases that regulate ion homeostasis in kidney epithelial cells where they promote uptake of Na+, K+ and Cl− ions from urine4. When activated, WNK1 phosphorylates and activates two related kinases OXSR1 and STK39, which in turn phosphorylate members of the SLC12A-family of electroneutral ion co-transporters, altering their activity. For example, phosphorylation of SLC12A2 (NKCC1) causes influx of Na+, K+ and Cl− ions, whereas phosphorylation of SLC12A6 (KCC3) blocks efflux of K+ and Cl− ions through the transporter5. Thus, signaling from WNK1 results in a net influx of ions.

We previously demonstrated that WNK1, the only WNK-family member expressed in mouse T cells, is required for the homing of CD4+ and CD8+ T cells to lymph nodes and spleen6. Furthermore, we showed that WNK1-deficient CD4+ T cells migrated more slowly in vivo in lymph node parenchyma6. WNK1-deficient T cells show increased integrin-mediated adhesion, which could have accounted for this reduced in vivo migration speed. However, blocking integrin adhesion further reduced in vivo migration speed, demonstrating that hyperadhesion was not the cause of reduced migration. We also showed that WNK1-deficient T cells have defective chemokine-induced migration in vitro6. We demonstrated that stimulation of CD4+ T cells with CCL21 results in rapid WNK1-dependent phosphorylation of OXSR1, which also required PI3K, indicating that CCR7 transduces signals via PI3K leading to WNK1 activation. Furthermore, CCL21-induced migration was partially inhibited in CD4+ T cells with mutations in Oxsr1 and Slc12a2 or treated with bumetanide (SLC12A2i), an SLC12A2 inhibitor6. Taken together, these results lead to the surprising conclusion that CCR7 signaling via WNK1 results in ion influx via SLC12A2 which is required for T cell migration. Thus, a pathway regulating ion homeostasis plays a critical role in T cell movement. However, it remains unclear why ion influx is required for this process.

Here we demonstrate that WNK1 pathway proteins and their activities are polarized to the leading edge of migrating T cells. Furthermore, we show that chemokine receptor-induced activation of the WNK1 pathway leads to ion influx and to water influx, with the latter most likely occurring via AQP3. We provide evidence that water ingress is required for actin polymerization and migration. We propose that this chemokine receptor-induced water entry generates increased spacing between the plasma membrane and the underlying actin cytoskeleton at the leading edge, which could facilitate the extension of actin filaments, and hence forward migration.

Results
WNK1 pathway proteins are required for CCL21-induced migration of CD4+ T cells

To understand how WNK1 pathway-regulated ion influx contributes to migration, we analyzed the functional requirement for the pathway proteins in T cells migrating in confinement, to model their migration within lymphoid tissue. Since constitutive loss of WNK1 in mice results in embryonic lethality7 and T cell lineage-selective loss results in a strong developmental arrest in the thymus with the absence of any mature WNK1-deficient T cells8, we chose to inducibly delete the Wnk1 gene in mature naive T cells, as previously described6. We crossed mice bearing a loxP-flanked Wnk1 allele (Wnk1fl) or a deletion of Wnk1 (Wnk1−) to mice with a tamoxifen-inducible Cre recombinase under the control of the ubiquitously expressed ROSA26 promoter (ROSA26CreERT2, RCE) to generate Wnk1+/flRCE and Wnk1fl/flRCE mice. Bone marrow from these animals was used to reconstitute the hematopoietic system of irradiated RAG1-deficient mice (Supplementary Fig. 1a). Subsequent treatment of these radiation chimeras with tamoxifen resulted in the generation of mice containing WNK1-expressing (Wnk1+/−RCE) and WNK1-deficient (Wnk1−/−RCE) T cells from which we isolated naive (CD25−CD44−) CD4+ T cells for further study.

We imaged the CCL21-induced migration of these CD4+ T cells under agarose on dishes coated with the integrin ligand ICAM-1. We found that in the absence of WNK1, T cells migrated more slowly (Supplementary Movie 1, Supplementary Fig. 2a, b, Fig. 1a). Moreover, whereas WNK1-expressing cells had a typical extended polarized morphology of migrating cells, WNK1-deficient cells showed no obvious polarization as measured by increased circularity (Supplementary Movie 1, Supplementary Fig. 2a–c). To evaluate the function of the WNK1 substrates OXSR1 and STK39 in T cell migration, we generated T cells mutated for both kinases, to account for any potential redundancy between them (Supplementary Fig. 1b). Using a tamoxifen-inducible ablation of Oxsr1, we found that CD4+ T cells deficient in OXSR1 and expressing a mutant STK39-T243A that cannot be phosphorylated and activated by WNK19 (Oxsr1−/−Stk39T243A/T243ARCE) also migrated more slowly in response to CCL21 in the same under agarose migration assay (Fig. 1b).

To extend this, we used an inhibitor against WNK1. WNK463 (WNKi), a selective WNK inhibitor10, completely blocked CCL21-induced phosphorylation of OXSR1 on S325, a known target of WNK1 activity11 (Supplementary Fig. 2d). WNKi showed a dose-dependent inhibition of CCL21-induced migration speed and shape polarization, with the extended cellular morphology of migrating T cells becoming more circular at higher WNKi doses (Supplementary Fig. 2e, f, Fig. 1c). 5µM WNKi resulted in a maximal inhibition of CCL21-induced migration and polarization, 0.5µM WNKi partially inhibited migration and polarization, whereas 0.2µM WNK1 reduced migration speed by a small amount but had no effect on polarization.

To analyze the role of the SLC12A-family of ion co-transporters, which are direct phosphorylation targets of OXSR1 and STK39 and at least five of which are expressed in CD4+ T cells (Supplementary Fig. 2g), we made use of both genetic mutation and an inhibitor. SLC12A2-deficient CD4+ T cells migrated more slowly in response to CCL21 compared to control SLC12A2-expressing cells, albeit the reduction in speed was smaller than that seen in WNK1-deficient cells (Fig. 1d). Furthermore, treatment of T cells with bumetanide, an SLC12A2 inhibitor that blocks ion movement (SLC12A2i), also caused decreased CCL21-induced migration speed (Fig. 1e). Polarization of the cell, however, was not affected (Supplementary Fig. 2h). These results support a role for the SLC12A2 ion co-transporter in T cell migration.

We extended these studies to CCL21-induced migration of CD4+ T cells in 3D in a collagen-I matrix. Again, naive WNK1-deficient CD4+ T cells, or T cells treated with WNKi or SLC12A2i showed reduced speeds of migration (Supplementary Movie 2, Supplementary Fig. 3a–c). Lastly, we asked if the requirement for WNK1 in chemokine-induced migration also applied to activated CD4+ T cells. We found that CXCL12-induced migration of activated CD4+ T cells under agarose was strongly reduced in the absence of WNK1, or by its inhibition (Supplementary Fig. 2i). Furthermore, in a collagen-I matrix, CXCL12-induced chemotaxis (directional movement towards the chemokine in a chemokine gradient) of activated CD4+ T cells was reduced by inhibition of WNK1 (Supplementary Fig. 2j). Thus, like naive CD4+ T cells, activated T cells also require WNK1 for chemokine-induced migration. Taken together, these results show that WNK1, OXSR1, STK39 and ion influx through SLC12A2 are required for CCL21-induced migration of T cells in confinement.

WNK1-regulated ion movement and its requirement for CD4+ T cell migration

The above results predict that CCL21 stimulation activates the WNK1 pathway, resulting in Na+, K+ and Cl− ion entry into the T cell, and that these ions are required for migration. To directly determine if chemokine stimulation induces ion movement in T cells, we used inductively-coupled plasma mass spectrometry (ICP-MS) to measure element concentrations in cell lysates and used these as a proxy for the abundance of ions. This showed that treatment with CCL21 resulted in an increase in K+ ions (Fig. 1f). Inhibition with WNKi reduced the amount of K+ in the T cells and blocked the CCL21-induced increase. It was not possible to reliably measure influx of Na+ or Cl− ions into the cells for technical reasons relating to high amounts of these ions in the extracellular medium. Thus, CCL21 stimulation activates a WNK1-dependent pathway that results in K+ entry into T cells, presumably in part through SLC12A2 or other SLC12A-family ion co-transporters.

To directly investigate if ions are required for T cell migration, we reduced the concentration of Na+ and Cl− ions in the medium while maintaining isotonicity. CCL21-induced migration of CD4+ T cells was substantially reduced as the concentrations of Na+ and Cl− ions were decreased (Fig. 1g). A similar effect was seen if we replaced Cl− ions with gluconate (Fig. 1h). Taken together, these results show that CCL21 stimulation results in WNK1-dependent ion entry into T cells, and that Na+ and Cl− ions are required for migration.

WNK1 pathway proteins regulate water entry and cell volume

WNK1-induced ion influx would be predicted to cause water entry by osmosis. In support of this, WNK1 has been shown to regulate the volume of non-lymphoid cells, allowing rapid responses to changing extracellular osmolarity by controlling movement of ions and hence water across the plasma membrane12. Thus, we hypothesized that in response to CCL21, WNK1-mediated ion influx would cause water entry, which may be required for migration. To determine if the WNK1 pathway regulates water movement in CD4+ T cells, we first measured the relative volume of cells with defects in the pathway using a Coulter principle-based cell counter. CCL21 induced a volume increase in WNK1-expressing T cells, whereas no such increase was seen in the absence of WNK1, or if WNK1 was inhibited (Fig. 2a, b). Furthermore, WNK1-deficient or WNK1-inhibited T cells were about 15% smaller even in the absence of CCL21, indicating a basal homeostatic function for WNK1 in regulating cell volume. It is likely that WNK1 is required for regulatory volume increase, a mechanism by which cells recover in volume after exposure to a hypertonic environment4. Mutation of Oxsr1 and Stk39 also caused T cells to become smaller and to no longer respond to CCL21 by increasing in volume (Fig. 2c). In contrast, SLC12A2i did not change basal T cell volume, but blocked the CCL21-induced volume increase (Fig. 2d), consistent with this inhibitor having the smallest effect on migration speed (Fig. 1e). To extend this analysis, we measured cell volume by imaging T cells migrating in response to CCL21. This showed that CCL21 induced a volume increase which was blocked by treatment with WNKi, and that the inhibitor also caused a decrease in basal T cell volume, consistent with the Coulter counter data, and in agreement with a role for WNK1 in homeostatic volume regulation (Fig. 2e, f).

To directly evaluate if chemokine stimulation results in water entry into CD4+ T cells, we used NMR to measure uptake of 2H2O into the cells. Similar to the volume analysis, CCL21 stimulation resulted in an increase in 2H2O in the cell, which was blocked by treatment with WNKi (Fig. 2g). Taken together, these results demonstrate that WNK1, OXSR1, STK39 and SLC12A2 regulate the volume of T cells in response to CCL21 stimulation, and that WNK1 regulates CCL21-induced water entry.

AQP3 is required for CCL21-induced T cell migration

Water enters cells through aquaporin (AQP) channels, a family of 11 proteins in the mouse. RNA-seq analysis shows that naive CD4+ T cells predominantly express Aqp3 and lower levels of Aqp9 and Aqp11 (Fig. 2h). To investigate the importance of water movement for T cell migration we used DFP00173 (AQP3i), a selective AQP3 inhibitor13. DFP00173 inhibits AQP3, but not AQP7 or AQP9; it is not known if it inhibits AQP11. Notably, AQP3i treatment caused a significant decrease in the speed of CCL21-induced migration of T cells under agarose or in a collagen-I matrix, in agreement with previous studies of AQP3-deficient T cells14 (Fig. 2i, Supplementary Fig. 3c). Treatment with AQP3i also reduced CCL21-induced polarization as measured by an increase in circularity (Supplementary Fig. 2h). Measurement of cell volume showed that like SLC12A2i, AQP3i did not change the basal volume of T cells but eliminated the CCL21-induced volume increase (Fig. 2d). A previous study had also proposed a role for AQP4 in T cell trafficking15. Using AER-270 (AQP4i) an AQP4 inhibitor16, we were able to show that treatment with this compound also reduced CCL21-induced migration speed, despite a lack of Aqp4 expression in the RNA-seq data (Fig. 2h, i). We conclude that either AQP4 protein is indeed expressed despite undetectable levels of Aqp4 mRNA, or that the AQP4i has off-target effects, perhaps on other AQPs.

AQP3 is a channel for H2O2 as well as water17. A previous study suggested that AQP3 is required for T cell migration because it facilitates H2O2 influx14. To investigate if H2O2 is required for mouse naive CD4+ T cell migration, we treated T cells with 20 U/ml catalase, a concentration sufficient to eliminate exogenously added 1mM H2O2 (Supplementary Fig. 4a). Treatment of T cells with this concentration of catalase did not impair migration in response to CCL21 (Supplementary Fig. 4b), implying that H2O2 is not required for T cell migration. It is not clear why our results differ from the earlier publication. However, we note several methodological differences. Whereas we directly measured migration speed in response to CCL21 under agarose, the earlier publication measured % migration through a Transwell in response to CXCL12. Furthermore, cells were incubated with 2000 U/ml catalase, 100-fold more than the concentration we used, which may have off-target effects. Taken together, these results show that AQP3 is required for CCL21-induced migration in CD4+ T cells, likely as a result of water transport, supporting the hypothesis that WNK1-regulated water entry is required for chemokine-induced T cell migration.

WNK1 pathway proteins polarize to the leading edge of migrating CD4+ T cells

To further understand how WNK pathway proteins may regulate chemokine-induced migration, we evaluated the sub-cellular distribution of the proteins and their activities in CD4+ T cells migrating in confinement under agarose. Live cell imaging of T cells expressing GFP-tagged WNK1, OXSR1 and SLC12A2 migrating in response to CCL21 showed that all three proteins were more abundant at the leading edge of the cell compared to the distribution of GFP only (Supplementary Movie 3, Fig. 3a–c). This accumulation at the leading edge was not a function of nuclear exclusion or passive enrichment at the leading edge of the fusion proteins, since nuclear-excluded GFP was enriched in the trailing part of the cell (Fig. 3d–f). Furthermore, imaging of fixed cells demonstrated that in migrating T cells, OXSR1, SLC12A2 and AQP3 are enriched at the leading edge and depleted at the trailing edge as identified by localization of CDC42 and CD44, respectively (Fig. 3g, h, Supplementary Fig. 5a, b). To analyze activity of the pathway we imaged the distribution of phosphorylated WNK1 (S382), OXSR1 (S325), and SLC12A2 (T203/T207/T212), phosphorylations that indicate increased activity of these proteins11,18,19. Notably, these phospho-proteins were also enriched at the leading edge of migrating T cells (Fig. 3g, h, Supplementary Fig. 5a, b). The polarization of these WNK1 pathway proteins and their activities was eliminated in WNK1-deficient or WNK1-inhibited cells (Supplementary Fig. 6a–j). Thus, in T cells migrating in response to CCL21, WNK1 pathway proteins and their activities accumulate at the leading edge of the cell, implying that CCR7 signaling via WNK1, OXSR1 and STK39 is likely to lead to localized entry of Na+, K+ and Cl− ions via SLC12A2 at the leading edge of the migrating cells.

The WNK1 pathway regulates spacing between the plasma membrane and the F-actin cytoskeleton

Migration of T cells requires actin polymerization at the leading edge which results in F-actin filaments pushing forward against the plasma membrane3,20. To determine if this process is controlled by the WNK1 pathway, we measured total F-actin in CCL21-stimulated CD4+ T cells using flow cytometry. This showed a rapid CCL21-induced increase in F-actin which was partially decreased by inhibition of WNK1 and SLC12A2 and by Oxsr1 and Stk39 mutations (Supplementary Fig. 7a–c), implying that the WNK1 pathway and ion influx may regulate actin polymerization.

Forward protrusion of actin filaments at the leading edge is generated by G-actin monomer addition to F-actin filament tips, most likely through a Brownian ratchet mechanism21–23. Thermal (Brownian) motion of both the membrane and the filaments results in a fluctuating gap, and when this exceeds 2.7 nm, an actin monomer can be added to the filament tip, thereby extending it. This prevents the membrane from returning to its original position, resulting in incremental forward protrusion. We hypothesized that WNK1-induced water entry at the leading edge would result in localized swelling of the membrane, pulling it away from underlying F-actin, thereby creating space into which actin filaments can polymerize, a mechanism we term a facilitated Brownian ratchet. To directly test if the WNK1-induced water movement affects spacing between the plasma membrane and the underlying F-actin, we first used instant structured illumination microscopy, a super-resolution method, to image the plasma membrane and F-actin in CD4+ T cells migrating in response to CCL2124. We found that at the leading edge the distance from the plasma membrane to the peak F-actin signal was around 174 nm, whereas at the side of the cell, this distance was around 43 nm, consistent with an increased spacing at the leading edge (Fig. 4a, b), although we note that this increased spacing could also be caused by a wider band of F-actin at the leading edge. Notably, treatment of the T cells with SLC12A2i or AQP3i caused a significant decrease in the membrane to peak F-actin spacing, with AQP3i again having the stronger effect (Supplementary Movie 4, Fig. 4c, d). Importantly, we noted a significant correlation between membrane to peak F-actin spacing and cell speed, supporting our hypothesis that this spacing is critical for T cell migration (Fig. 4e). Thus, our data suggest that SLC12A2 and AQP3 function are required to maintain a larger membrane to peak F-actin spacing at the leading edge and are consistent with water entry causing increased spacing between the membrane and the underlying F-actin. However, we cannot rule out that the changes in these distances are caused by an altered width of cortical F-actin in the migrating cell which could also correlate with migration speed.

WNK1 activity and ion and water influx regulate membrane proximal F-actin at the leading edge

Surprisingly, analysis of F-actin in migrating cells has shown that despite greatly elevated levels of F-actin at the front of the cell, there is a lower density of membrane-proximal actin (MPA) within 10 nm of the leading edge compared to other parts of the migrating cell25. This is possibly due to a reduced load of the membrane on actin filaments at the leading edge, which results in rapid polymerization of actin filaments perpendicular to the membrane. These outcompete a network of filaments at shallow angles, thereby generating a region with lower F-actin density proximal to the plasma membrane26. We hypothesized that WNK1-induced water entry and membrane swelling may contribute to the reduced load and hence lower MPA at the leading edge. To test this, we analyzed migrating T cells expressing two fluorescent probes: MPAct-mCherry (MPAct) and GFP-CaaX (CaaX)25. Both contain fluorescent proteins that are tethered to the plasma membrane, but MPAct also contains an F-actin-binding domain. Thus, CaaX is evenly distributed on the plasma membrane, whereas MPAct accumulates in regions of the membrane with higher concentrations of underlying MPA that is within 10 nm of the membrane. Hence the MPAct/CaaX ratio is a measure of the distribution of MPA. Analysis of T cells expressing MPAct and CaaX migrating in response to CCL21, showed that, as expected, the MPAct/CaaX ratio is reduced at the leading edge compared to the side and trailing edges (Fig. 5a, b). Notably, treatment of T cells with WNKi, SLC12A2i or AQP3i resulted in a significant increase in MPA at the leading edge, with less polarization of the MPA signal between the front and back of the cell (Supplementary Movie 5, Supplementary Fig. 7d, e, Fig. 5c–f). Importantly, for these studies we used a low dose of WNKi (0.2 µM) which reduces migration speed but still allows cells to polarize (Supplementary Fig. 2e). Thus, the activities of WNK1, SLC12A2 and AQP3 are required to maintain reduced MPA at the leading edge of T cells, consistent with our hypothesis that localized ion influx and subsequent water entry causes the membrane to swell away from the underlying F-actin.

WNK1 pathway regulates actin retrograde flow in migrating T cells

If WNK1-induced water entry at the leading edge is required to generate space and hence facilitate the Brownian ratchet mechanism of actin filament growth, inhibition of the pathway should affect the rate of actin polymerization at the membrane. Continuous addition of G-actin monomers at the leading edge of migrating cells results in retrograde flow of F-actin, the rate of which can thus be used as a surrogate for actin polymerization rate at the front of the cell27. To determine if the WNK1 pathway regulates this process, we measured rates of retrograde actin flow in CCL21-stimulated T cells expressing LifeAct-eGFP to visualize F-actin. The cells were imaged responding to CCL21 on PEG-coated glass, an arrangement that eliminates integrin-based adhesion, causing the cell to stay in place and the F-actin cytoskeleton to slip in relation to the substrate. In contrast to a partial decrease in total F-actin, retrograde actin flow was strongly decreased in WNK1-deficient or WNK1-inhibited T cells (Supplementary Movie 6, Supplementary Fig. 8a, b, Fig. 6a, b). Furthermore, both SLC12A2i and AQP3i partially slowed retrograde actin flow, with AQP3i having the stronger effect. Thus, WNK1, SLC12A2 and AQP3 function are required for actin retrograde flow, consistent with our hypothesis that ion and water influx influence the rate of addition of G-actin monomers to the growing tips of F-actin filaments at the leading edge.

WNK1-induced water entry is required for T cell migration

To directly determine if WNK1-induced water entry is required for migration, we asked if forcing water into the cells by reducing the tonicity of the medium could rescue migration in WNK1-inhibited cells. This volume increase in response to hypotonic medium would counteract the defective regulatory volume increase observed in the absence of WNK1. For these studies we again used a low dose of WNKi to slow the cells down, but still allow them to polarize. Remarkably, while reducing tonicity caused a reduction of migration speed in T cells treated with vehicle control, it increased migration speed in cells where WNK1 had been partially inhibited. This was most effective at 248 mOsm/l, a ~15% reduction of tonicity compared to isotonic medium (292 mOsm/l) (Fig. 6c). The hypotonic rescue of migration in WNK1-inhibited T cells could be due to the increased water entry, or, alternatively, a result of reduced NaCl concentration. For example, since Cl− is an inhibitor of WNK112,28, it is possible that the reduced Cl− concentration in the hypotonic medium could lead to reduced intracellular Cl− thereby increasing WNK1 activity and overcoming the effect of a low dose (0.2 µM) of WNKi. To distinguish if the rescue of migration was caused by increased water entry or decreased NaCl, we compared the migration speed of WNK1-inhibited T cells in hypotonic medium at 248 mOsm/l or in isotonic medium with the same reduced amount of NaCl but with isotonic osmolarity restored using L-glucose, an inert osmolyte (Fig. 6d). Whereas hypotonic medium rescued most of the migration defect of WNK1-inhibited cells, isotonic medium with reduced NaCl was less effective, demonstrating that water entry is required for T cell migration. One caveat to this conclusion is that, depending on the chloride conductance of the cell, the isotonic medium with reduced NaCl may result in less reduction of intracellular chloride compared to the hypotonic medium.

In further experiments, we found that treatment of WNK1-inhibited cells with hypotonic medium also restored lower MPA levels at the leading edge of migrating cells (Fig. 5c, e). Finally, the reduced retrograde actin flow in WNK1-inhibited T cells was partially rescued when the cells were placed in hypotonic medium (Fig. 6e). Taken together, these results support our hypothesis that WNK1-induced osmotic water entry is required for CCL21-induced actin retrograde flow and T cell migration.

Methods
Mice
Mice with a conditional allele of Wnk1 containing loxP sites flanking exon 2 (Wnk1tm1Clhu, Wnk1fl)7, with a deletion of exon 2 of Wnk1 (Wnk1tm1.1Clhu, Wnk1−)6, with a conditional allele of Oxsr1 containing loxP sites flanking exons 9 and 10 (Oxsr1tm1.1Ssy, Oxsr1fl)41, with a tamoxifen-inducible Cre in the ROSA26 locus (Gt(ROSA)26Sortm1(Cre/ESR1)Thl, ROSA26CreERT2, RCE)42, with an allele of Stk39 encoding STK39-T243A (Stk39tm1.1Arte, Stk39T243A)9 that cannot be activated by WNK1, expressing LifeAct-eGFP (Tg(CAG-EGFP)#Rows)43, expressing a LifeAct-eGFP tdTomato protein from the ROSA26 locus (Gt(ROSA)26Sortm4(ACTB-tdTomato-EGFP)Luo, ROSA26-mTmG)44 or deficient for SLC12A2 (Slc12a2tm1Ges, Slc12a−)45 or RAG1 (Rag1tm1Mom, Rag1−)46 have been described before. C57BL/6J mice were provided by the Biological Research Facility of the Francis Crick Institute. All mice were maintained on a C57BL/6J background and intercrossed as required to generate strains with multiple genetically altered alleles. Mice were kept in rooms with controlled 12 h light/dark cycles at 23 °C and 50% humidity. Mice were housed in individually ventilated cages under specific pathogen-free conditions, and given food and water ad libitum. Both female and male mice were used and were at least 6 weeks old at the time of the experiment. All mice were age- and sex-matched within an experiment. All experiments were approved by the Francis Crick Institute Animal Welfare Ethical Review Board and were carried out under the authority of a Project Licence granted by the UK Home Office.

Bone marrow chimeras
Bone marrow cells from Wnk1fl/+RCE and Wnk1fl/−RCE mice or from LifeAct-eGFPWnk1fl/+RCE and LifeAct-eGFPWnk1fl/−RCE mice or from Oxsr1+/+Stk39+/+RCE, Oxsr1fl/flStk39+/+RCE, and Oxsr1fl/fl/Stk39T234A/T234ARCE mice were transferred intravenously into irradiated (5 Gy, 137Cs source) RAG1-deficient recipient mice which were maintained on 0.02% enrofloxacin (Baytril, Bayer Healthcare) for 4 weeks. In all cases the sex of the donor and recipient mice was matched. The hematopoietic compartment was allowed to reconstitute for at least 8 weeks after bone marrow transfer. In all experiments, control and experimental chimeric mice or cells derived from them were always sex-matched.

In vivo deletion of floxed alleles
Tamoxifen treatment of Wnk1fl/+RCE and Wnk1fl/−RCE bone marrow chimeras was used to generate Wnk1+/−RCE and Wnk1−/−RCE T cells. Tamoxifen treatment of Oxsr1+/+Stk39+/+RCE, Oxsr1fl/flStk39+/+RCE, and Oxsr1fl/fl/Stk39T234A/T234ARCE bone marrow chimeras was used to generate Oxsr1+/+Stk39+/+RCE, Oxsr1−/−Stk39+/+RCE, and Oxsr1−/−/Stk39T234A/T234ARCET cells, respectively. Mice were injected intraperitoneally with 2 mg tamoxifen in corn oil (Sigma-Aldrich) on 3 (Wnk1fl) or 5 (Oxsr1fl) consecutive days. Mice were sacrificed 7 (Wnk1fl) or 21 (Oxsr1fl) days after initial tamoxifen injection to harvest T cells.

T cell purification and culture
To isolate naïve CD4+ T cells, mouse lymph node cells were incubated with biotin-conjugated antibodies to CD44 (IM7) and CD25 (PC61.5) (both from ThermoFisher, both at 1:400 dilution), CD11c (N418), Ly6G (RB6-8C5), CD8α (53-6.7), B220 (RA3-6B2) and TER119 (TER-119) (all from BioLegend) and CD19 (1D3, BD Biosciences) (all at 1:200 dilution), and antibody-bound cells were removed with streptavidin-coated Dynabeads (ThermoFisher) using magnetic-activated cell sorting according to the manufacturer’s instructions. Isolated T cells were rested overnight at 37 °C and 5% CO2 in R10 medium, which consisted of RPMI 1640 (Sigma) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, non-essential amino acids, 100 µM 2-mercaptoethanol (all Sigma-Aldrich) and 20 mM HEPES (Gibco).

T cell migration under agarose
8-well polymer or glass chamber µ-slides (ibidi, 80821 or 80827) were coated with recombinant mouse ICAM-1-Human-Fc chimera (RnD, 796-IC) at 3 µg/ml in PBS overnight at 4 °C. Slides were washed 3x with PBS, blocked with 2% bovine serum albumin (BSA) (Sigma-Aldrich), and washed 3x again. Agarose gel preparation was adapted from a previously described method47. Agarose (0.5%, UltraPure Agarose, ThermoFisher) in R10-equivalent medium was prepared at 50 °C, and, if required, mouse CCL21 (250 ng/mL RnD, 457-6C) and inhibitors [WNK463, (WNKi, MedChemExpress), bumetanide (SLC12Ai, 20 µM, Abcam), DFP00173 (AQP3i, 20 µM, Axon MedChem), AER-270 (AQP4i, 0.25 µM, Tocris)] or equivalent amounts of vehicle control (DMSO, ThermoFisher) were added, and the mixture immediately plated onto the µ-slides. The agarose was cooled at 4 °C for 1 h, and then equilibrated at 37 °C and 5% CO2 for at least 30 min. To change the osmolarity of the gel, part of the solution was replaced with NaCl-free HBSS to generate a gel of the required osmolarity. For isotonic agarose gels with reduced NaCl, the NaCl-free HBSS was supplemented with L-glucose (Sigma). Osmolarity was verified using an osmometer (Model 3250, Advanced Instruments).

T cells stained with CellTraceViolet (CTV, 1 µM, ThermoFisher) were resuspended in a small volume of R10 medium containing propidium iodide (1.5 µM) and injected under the agarose gel with a pipette. Cells were acclimatized at the microscope for at least 30 min in a humidified chamber at 37 °C and 5% CO2 and then imaged every 15 s for 10 min using a Nikon Ti2 Eclipse microscope with an LED illumination system. Images were acquired with a Nikon 20x (0.75 NA) Ph2 plan apo objective, at 2048 × 2048 resolution in 16-bit using Micro-Manager48. Cells were tracked using the FIJI plugin TrackMate49. Propidium iodide-containing dead cells, and tracks shorter than half the video length were excluded. To determine cell shapes, binary images were produced from the CTV signal from one still frame per video stack using the FIJI ‘triangle’ auto-thresholding algorithm. Cell profiles were smoothed using the ‘open’ binary function and particles between 38 and 60 µm2 were detected with the ‘analyze particle’ tool.

T cell migration in a Collagen-I matrix
To study T cell migration in a collagen-I matrix a previously described method was adapted50. CTV-labeled CD4+ T cells (2 × 106 cells/ml) in R10 medium containing rat tail Collagen-I (1.5 mg/ml, ibidi) and CCL21 (250 ng/ml) were mixed on ice and plated into wells of a 15-well angiogenesis µ-slide (ibidi) and incubated at 37 °C and 5% CO2 for 30 min to allow the collagen to polymerize. R10 medium containing CCL21, propidium iodide (1.5 µM) and inhibitors or vehicle control if required, was added to the wells and slides incubated in a humidified chamber on the microscope at 37 °C and 5% CO2 for a further 30 min. Cells were imaged using a Zeiss LSM880 with an AiryScan detector, a 405 nm laser to excite both CTV and propidium iodide and a Zeiss 10x plan apo (0.45 N.A.) air objective.

A z-stack consisting of 14 z-slices with an 8 µm spacing were acquired every 15 s for 5–10 min. Cells were tracked using the FIJI plugin TrackMate. Propidium iodide-positive cells and cells that were tracked for less than 1/3 of the video length were excluded from the analysis.

Migration of activated T cells
Flat-bottomed multi-well cell culture plates (Corning) were incubated overnight at 4 °C with PBS containing plate-bound anti-CD3ε (4 µg/ml, 145-2C11, Tonbo Biosciences) and anti-CD28 (4 µg/ml, 37.51, BioLegend). Plates were washed with PBS and mouse naïve CD4+ T cells were cultured in the wells at 1 × 106 cells/ml in R10 at 37 °C. On day 3 of activation, cells were transferred into fresh plates, stimulated daily with 20 ng/mL recombinant IL-2 (PeproTech) and used on day 5 of activation. Under agarose migration assays were run as described above, except using 250 ng/mL CXCL12 (R&D Systems, 460-SD-01/CF) instead of CCL21.

Chemotaxis assays were run according to the manufacturer’s instructions (ibidi µ-slide chemotaxis, 80326) using CXCL12 as a chemoattractant. Briefly, cells were suspended in a 3D 1.5 mg/mL rat-tail collagen-I matrix in the observation area of the slide. Chemotaxis chambers reservoirs were loaded and cells were imaged for 1 h using a Nikon Ti2 Eclipse and a 20x (0.75 NA) Ph2 objective at 37 °C and 5% CO2.

Immunoblotting
Mouse naïve CD4+ cells were rested in T cell media for >3 h. 2–4 × 106 cells per condition were preincubated with vehicle control or WNKi for 30 min at 37 °C. Cells were stimulated with 250 ng/mL CCL21 for the indicated amount of time. Cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 50 mM NaF, 5 mM dithiothreitol, 2 mM EDTA, 2 mM Na4P2O7, 1 mM Na3VO4, 1% Triton X100, 0.5% deoxycholate, 0.1%, sodium dodecyl sulfate (SDS), 5 mM dithiothreitol, 1× PhoStop (Roche), 1× complete protease inhibitor cocktail (Roche)) for 15 min on ice. Samples were pelleted at 16,000 × G for 10 min at 4 °C. Lysates were diluted in SDS sample buffer (0.2 M Tris-HCl, 3% SDS, 10% glycerol, 3% 2-mercaptoethanol, 0.25% bromophenol blue) and heated at 95 °C for 5 min.

Lysates were analyzed by electrophoresis on 4–20% SDS-PAGE gels at 120 V for 1 h in Tris-Glycine-SDS running buffer. Separated proteins were transferred to methanol-activated PVDF membranes using Turboblot transfer (BioRad). Membranes were blocked for 90 min in Li-Cor Odyssey Blocking buffer. Primary antibodies (anti-pOXSR1 polyclonal, MRC-PPU Dundee; anti-ERK2, Cell Signalling Technologies, 9108) were diluted in Li-Cor buffer at 1:500 dilution and incubated overnight at 4 °C. Membranes were washed 3x in PBS + 0.1% Tween 20 (PBST). Membranes were incubated with secondary antibodies [α-sheep IgG (H+L) AlexaFluor 680 and α-rabbit IgG (H+L) WesternDot 800 (both Invigrogen)] at 1:1000 at room temperature for 1 h, washed in PBST and imaged using Li-Cor CLx. Blots were analyzed on Image Studio.

Determination of element concentrations using inductively-coupled plasma mass spectrometry (ICP-MS)
ICP-MS samples were prepared using a protocol adapted from Stangherlin et al.51. T cells were rested overnight in R10 medium. Cells were pre-treated with inhibitor or vehicle in R10 for at least 20 min at 37 °C and 5% CO2. Following pre-treatment, cells were stimulated with CCL21 (250 ng/mL final concentration) or medium only for 10 min at 37 °C and 5% CO2. Cells were washed twice in ice-cold iso-osmotic wash buffer (300 mM sucrose, 10 mM Tris base, 1 mM EDTA in purified water, pH 7.4 adjusted with acetic acid [Fluka]). Cell pellets were digested in trace metal grade concentrated nitric acid (67–69% w/w) for 15 min at room temperature, diluted using purified water with a resistivity ≥ 18.2 MΩ cm from a Milli-Q system (Merck Millipore) and then spiked with either gallium (Ga) or cerium (Ce). Sample measurements were conducted at two institutes. At the London Metallomics Facility at King’s College London, samples were analyzed on a Thermo Scientific iCAP TQ Inductively Coupled Plasma Quadrupole Mass Spectrometer (ICP-QMS) operating in Dynamic Reaction Cell (DRC) mode with oxygen as the reaction gas. The introduction system to the instrument was a Cetac ASX-520 autosampler coupled to a MicroMist nebulizer that was fitted to a quartz cyclonic spray chamber. Data reduction involved the normalization of raw intensities using Ga as an internal standard, then blank correcting the signal by removing the average analyte intensity of repeat blank measurements and applying external standardization using a six-point calibration curve to convert the corrected intensities into concentration measurements. At the Department of Earth Sciences, University of Cambridge, samples were analyzed using a PerkinElmer Nexion 350D ICP-MS and analyte intensities were normalized based on cerium (Ce) (Romil, E3CE#). For each sample, element concentrations were normalized to cell numbers. Data from the two measurement sites were combined by normalizing element concentrations to unstimulated control cells.

NaCl reduction and Cl replacement
To generate media of varying NaCl compositions, NaCl-free RPMI was supplemented with additional NaCl (Honeywell, S9888). To make the medium isotonic, the remaining difference in osmolarity (up to 300 mOsm/L) was made up using D-sorbitol (Sigma, S1876), an inert osmolyte. Osmolarity was verified using an osmometer (Model 3250, Advanced Instruments). To generate medium with reduced Cl concentration, NaCl-free RPMI was made isotonic through the addition of Na gluconate (Sigma, S2054-100G). Media was supplemented with 0.5% BSA and propidium iodide (1.5 µM). CTV-stained T cells were suspended in medium containing 250 ng/mL CCL21, allowed to adhere for 1 h at 37 °C and imaged on a Nikon Ti2 Eclipse widefield microscope using a 20x (0.75 NA) Ph2 objective at 37 °C and 5% CO2.

Cell volume measurement
T cells were rested overnight in R10 medium. Inhibitor or vehicle control was added for 20 min and then CCL21 or medium for a further 20 min. Cells were resuspended in CASYton fluid (Cambridge Bioscience) and median cell volumes measured using the Coulter counter principle on a CASY cell counter (Cambridge Bioscience).

Alternatively, cell volume was determined using microscopy. Naïve CD4+ T cells purified from ROSA-mTmG mice expressing tdTomato-CAAX to visualize the plasma membrane were imaged in glass 8-well µ-slides (ibidi, 80827) coated with ICAM-1 (3 µg/mL) in phenol red-free RPMI supplemented with 10% FCS. Cells were treated with WNKi or vehicle control and stimulated with 250 ng/mL CCL21 and rested on the microscope for 30 min prior to imaging. Cells were imaged using instant structural illumination microscopy (iSIM) using an iSIM microscope (VisiTech) with an Olympus 150x TIRF apo (1.45 NA) oil-immersion objective. Cells were excited using 552 nm wavelength laser, with emission detected using an sCMOS camera (Prime BSI Express, Teledyne Photometrics). Cells were either imaged for 2 min every 15 s or acquired as a still frame. Resulting images were deconvoluted using Microvolution algorithms in µ-Manager. Cell volume was calculated by generating a mask of plasma membrane edges and summing up the areas of individual z-slices in each cell measured using the ‘analyze particles’ function in FIJI.

2H2O uptake assay
T cells were rested overnight in R10 medium. Cells from one biological replicate were divided into four conditions and pre-treated with WNKi or vehicle in H2O-based R10 for at least 20 min at 37 °C and 5% CO2. Following pre-treatment, cells were stimulated with 250 ng/mL CCL21 in 2H2O-based R10 (deuterium oxide, Sigma-Aldrich, 435767) containing vehicle or WNKi. Cells were washed twice in ice-cold H2O-based PBS and lysed using lysis buffer (50 mM HEPES, 150 mM NaCl, 1% NP40 in ultrapure H2O containing 1:500 uniformly 2H-labeled 1,4-dioxane-d8 [Sigma Aldrich, 186406] as an internal standard) for 20 min at room temperature. Lysate supernatants were measured using nuclear magnetic resonance (NMR) spectroscopy (Bruker Advance III HD 700 equipped with a 5 mm QCI cryoprobe) at 25 °C employing 3 mm NMR tubes. Fully-relaxed one-dimensional 2H spectra were recorded in the absence of field-frequency lock using a 30° excitation pulse, 8 s relaxation delay, 20 ppm sweep width, 1.6 s acquisition time, 2 dummy scans and 16 accumulated transitions (total experiment time 3 min per sample). Data processing in Bruker Topspin 3.6 employed zero-filling to 128K points, 4 Hz line-broadening, Fourier transformation and baseline correction. For each sample, the peak integral of the 2H resonance of 2H2O was normalized to that of the 2H-labeled 1,4-dioxane.

Catalase treatment
To determine the efficacy of catalase treatment, mouse naïve CD4+ T cells were loaded with DCFDA (ab113851, Abcam) according to the manufacturer’s protocol. Cells were pre-treated with bovine liver catalase (C40, Sigma Aldrich) for 30 min at the indicated concentrations. Cells were treated with 1 mM H2O2 for 30 min at room temperature, and DCFDA fluorescence was measured by flow cytometry (LSR Fortessa X-20, BD Biosciences).

For migration experiments, overnight rested mouse naïve CD4+ T cells were pre-treated with 20 U/mL catalase in R10 for at least 30 min. Cells were injected under agarose containing 20 U/mL catalase and CCL21 in 8-well dishes coated in ICAM-1, as described in other experiments. Cells were rested for 30 min at the microscope prior to imaging as described above.

Generation of CD4+ T cells expressing GFP-WNK1, GFP-OXSR1 and GFP-SLC12A2
DNA fragments encoding open reading frames for GFP-WNK1, GFP-OXSR1 and GFP-SLC12A2 were inserted into the MIGR1 retroviral vector52, replacing the IRES-GFP sequence, such that fusion proteins were expressed from the 5’ LTR of the vector. In all cases the eGFP was fused in-frame to the N-terminus of the human WNK pathway proteins. The GFP-WNK1 and GFP-SLC12A2 constructs were generated by the MRC Protein Phosphorylation Unit. All constructs were validated by DNA sequencing.

Plasmid DNA for MIGR1 vectors expressing GFP-WNK1, GFP-OXSR1 or GFP-SLC12A2 and MIGR1 expressing GFP only were transfected into PlatE cells53 to generate medium containing infectious retrovirus. This was used to infect bone marrow cells from C57BL/6J mice that were in turn used to reconstitute irradiated (5 Gy, 137Cs source) RAG1-deficient mice54.

Live cell imaging of T cells expressing WNK1 pathway proteins
CD4+ T cells expressing GFP-tagged WNK1 pathway proteins were purified from lymph nodes of bone marrow chimeras by flow cytometry sorting for GFP+ T cells using an Avalon sorter (Propel Labs/Bio-Rad). Sorted cells were incubated overnight in R10 medium at 37 °C and 5% CO2 and then injected under agarose in an 8-well plastic bottom µ-slide containing CCL21 as described earlier. Cells were placed in a humidified chamber at 37 °C and 5% CO2 on a Zeiss LSM880 inverted microscope for 30 min, and then imaged by confocal microscopy with a 63x plan apo (1.4 N.A.) oil-immersion objective. Cells were imaged every 15 s for 5 min in a single z-plane proximal to the coverslip. To determine the localization of GFP or GFP-tagged proteins in migrating T cells we used the FIJI plugin VirusTracker55. The plugin detects the direction of migration and was set to capture a 20 µm-long region of interest along the length of the cell. Images of these cell shapes were projected on top of each other using the mean intensity projection option.

To image nuclear-excluded GFP, mouse naïve CD4+ T cells were transfected with p8xEGFP-N1 (Addgene # 122168, gift from Georg Mayr) which expresses 8 concatemeric EGFP proteins, resulting in nuclear exclusion. Cells were electroporated using the X-001 program on a Nucleofector (Lonza) using a mouse T cell nucleofection kit (Lonza, VPA-1006), and rested overnight in R10 medium. EGFP+ T cells were sorted and incubated in R10 medium for at least 2 h at 37 °C and 5% CO2. Cells were injected under agarose containing CCL21 and allowed to acclimatize for at least 30 min in a humidified chamber on the microscope at 37 °C and 5% CO2. Cells were imaged using a Leica SP8 FALCON confocal inverted microscope, equipped with a 63x plan apo (1.4 NA) oil-immersion objective every 15 s for 2.5 or 5 min. Cells were excited using a white light source at 488 nm for EGFP. To determine protein localization, cells were aligned in the direction of migration and a 14.4 × 7.5 µm box was drawn around the outline of the cell. Images of these cell shapes were projected on top of each other using the mean intensity projection option.

Imaging of WNK1 pathway proteins in fixed T cells
15-well angiogenesis or 18-well µ-slides (ibidi, 81506 or 81816) were coated with 3 µg/ml mouse ICAM-1-human Fc fusion protein overnight at 4 °C. Slides were washed 3x with PBS, blocked with 2% BSA in PBS for 20 min at room temperature, and washed again three times. Naïve CD4+ T cells that had been incubated overnight at 37 °C were resuspended in RPMI containing 0.5% BSA, added to the µ-slides, and allowed to adhere for 1 h at 37 °C. Cells were stimulated with 250 ng/ml CCL21 for 20 min, and then fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were washed with PBS, blocked in 1% BSA in PBS (blocking buffer) for 20 min, permeabilized with 0.1% Tween 20 in PBS for 15 min, washed with PBS and stained in blocking buffer overnight at 4 °C with anti-CDC42 (B-8 mouse monoclonal, Santa Cruz), biotin-conjugated anti-CD44 (IM7 rat monoclonal, BioLegend) and one of these primary antibodies: anti-p-WNK1 (S382), anti-OXSR1, anti-p-OXSR1 (S325), anti-SLC12A2, anti-p-SLC12A2 (T203/T207/T212) (all sheep polyclonal sera from MRC Protein Phosphorylation Unit) or anti-AQP3 (NBP1-97927 rabbit polyclonal, Novus Biologicals), all diluted at 1:200. Cells were washed with PBS and incubated in blocking buffer for 1 h with Streptavidin-AlexaFluor 488 (S11223, Molecular Probes), goat anti-Mouse IgG-AlexaFluor 555 (A-21424), and either goat anti-rabbit IgG-AlexaFluor 647 (A-21245) or donkey anti-sheep IgG-AlexaFluor 647 (A-21244, all from ThermoFisher), all diluted at 1:250. Cells were washed twice with 0.1% Tween 20 in PBS and once with PBS containing DAPI to visualize the nucleus. Cells were washed once and covered with VectaMount AQ aqueous mounting medium (Vector Laboratories) and stored at 4 °C until imaging.

Cells were imaged on a Leica SP8 FALCON inverted confocal microscope with a 63× plan apo (1.4 NA) oil-immersion objective. Fluorophores were excited with a white light laser. Cells were imaged in z-stacks consisting of 7–9 z-slices with a 1 µm spacing. To determine localization of WNK1 pathway proteins, CDC42 and CD44 were used to determine the leading and trailing edges (LE and TE) respectively. A line the full width of the cell was drawn along the length of the cell from the LE to the TE. Average fluorescence intensities were determined for each of 12 bins along the cell length. Intensity profiles were normalized from 0–1, where 0 was the lowest signal and 1 the highest signal per protein, per cell. Binned normalized fluorescence intensities were used to calculate the correlations between LE and TE markers and WNK1 pathway proteins. To evaluate polarization of WNK1 pathway proteins, fluorescence was measured along the perimeter of the cell and the polarization ratio was calculated by dividing the fluorescence in the 25% of the perimeter centered on the maximum fluorescence by the total fluorescence along the perimeter of the cell.

Flow cytometric analysis of F-actin
Overnight-rested mouse naïve CD4+ T cells were placed in RPMI, 0.5% BSA and stimulated with CCL21 for the required time. Cells were immediately fixed in 4% paraformaldehyde for 15 min. Cells were washed with PBS, permeabilized with 0.1% Tween 20 in PBS for 15 min, and blocked for 1 h in 2% BSA in PBS. Cells were stained with phalloidin conjugated to iFluor-488 (Abcam) in blocking buffer for 1 h at room temperature, washed twice with 0.1% Tween 20, and once with PBS. Cells were analyzed on an LSRII flow cytometer (BD Biosciences) and mean fluorescence intensity was quantified using FlowJo (BD Biosciences).

Analysis of plasma membrane to F-actin spacing
Overnight-rested mouse naive CD4+ T cells constitutively expressing LifeAct-eGFP were labeled with CellMask Orange (ThermoFisher) to stain the plasma membrane following the manufacturer’s instructions. Cells were washed once with PBS, resuspended in R10 medium and injected under CCL21- and inhibitor-containing agarose in a glass bottom 8-well µ-slide dishes (ibidi) pre-coated with ICAM-1. Cells were imaged by iSIM using an iSIM microscope (VisiTech) with an Olympus 150x TIRF apo (1.45 NA) oil-immersion objective. Cells were simultaneously excited at 488 nm for LifeAct-eGFP and 561 nm for CellMask Orange. Emission fluorescence was split with a dichroic mirror with a long-pass filter at 561 nm and was simultaneously detected using 2 sCMOS cameras (Prime BSI Express, Teledyne Photometrics). Cells were imaged every 250 ms for a total of 30 s in a single z-plane proximal to the coverslip. To correct for chromatic aberration and camera alignment, 0.5 µm TetraSpeck fluorescent beads (ThermoFisher) were imaged after each session using the same acquisition settings as the cells.

Images were deconvoluted using Huygens software (Scientific Volume Imaging) with the ‘Normal’ deconvolution template. A random frame from the time-lapse series was chosen while viewing only the plasma membrane stain to avoid bias. The leading edge of the cell was determined from the direction of migration. Adapting a previously described analysis protocol56, a 30-pixel wide line was drawn along the leading edge, with the plasma membrane placed in the middle of the line. The line and the data within it were straightened out and 20-pixel wide overlapping lines were drawn perpendicular to the plasma membrane signal, along the length of the leading edge. Fluorescence intensity data were normalized from 0-1, and the intensity profiles in each 20-pixel wide line were manually aligned according to the peak of plasma membrane fluorescence intensity. The distance between the peak plasma membrane signal and the peak F-actin signal was calculated within each line, with the peak of F-actin defined as the highest LifeAct-eGFP fluorescence intensity within 400 nm of the plasma membrane.

Analysis of membrane proximal F-actin
Mouse naïve CD4+ T cells were transfected with C1-MPAct-mCherry (Addgene #155222, gift from Tobias Meyer) and C1-eGFP-CaaX (Addgene #86056, gift from Lei Lu) plasmids using the X-001 program on a Nucleofector (Lonza) and rested overnight in T cell medium. GFP+ mCherry+ T cells were sorted and incubated in R10 medium for at least 2 h at 37 °C and 5% CO2. Cells were injected under agarose containing CCL21 and inhibitors or vehicle control and allowed to acclimatize for at least 30 min in a humidified chamber on the microscope at 37 °C and 5% CO2. Cells were imaged using a Leica SP8 FALCON confocal inverted microscope, equipped with a 63x plan apo (1.4 NA) oil-immersion objective every 15 s for 2.5 or 5 min. Cells were excited using a white light source at 488 nm for eGFP-CaaX and at 585 nm for MPAct-mCherry, and acquired simultaneously.

To analyze the distribution of the MPAct probe, one random frame per migrating cell was selected using FIJI. Bias was avoided by selecting frames using only the GFP-CaaX signal. The leading edge of the cell was determined from the direction of migration. A line the full length and width of the cell was draw from the front to the back of the cell. Average fluorescence intensities were determined for each of 12 bins along the cell length. Fluorescence intensity levels were normalized with the highest intensity value per fluorophore, per cell set to 1. The normalized MPAct-mCherry signal was divided by the normalized eGFP-CaaX signal to generate the MPAct/CaaX ratio. Polarization of the MPAct signal was determined by dividing the MPAct/CaaX ratio in the front 3 bins by the ratio in the rear 3 bins.

TIRF microscopy of F-actin retrograde flow
35 mm round glass-bottom dishes (Greiner Bio-One) were cleaned with 1 M HCl for 15 min and washed three times with water. Dishes were plasma-activated for 30 s (HPT-100, Henniker Plasma) and coated with 0.2 mg/ml poly(L-lysine)-graft-PEG (SuSoS) in PBS overnight at 4 °C, blocked with 2% BSA in PBS for 20 min, and washed three times with PBS. Overnight-rested mouse naïve CD4+ T cells constitutively expressing LifeAct-eGFP were injected under agarose containing CCL21 and either inhibitors or vehicle control. Cells were imaged at 37 °C and 5% CO2 using an Olympus IX83 total internal reflection fluorescence (TIRF) microscope with a 150 × 1.45 NA TIRF apo oil-immersion objective every 1 s for 1 min. Actin flow rates were determined from kymographs generated using FIJI.

RNA sequencing data
Data on expression of Aqp and Slc12a mRNAs in mouse CD4+ T cells was taken from an RNA sequencing analysis of naive mouse CD4+ T cells (Sequence Read Archive, SRA accession number SRP059425)6.

Statistical analysis
All measurements were made on distinct samples (e.g. cells). No sample was measured repeatedly. To determine statistical significance between two unpaired groups, we used a 2-sided Mann–Whitney U test. To determine statistical significance when there were more than two groups and one variable, we used either a Kruskal-Wallis test when the data was not normally distributed, and unpaired and unmatched, or a 1-way ANOVA when the data was normally distributed and unmatched. When comparing multiple paired groups, we used a Friedman test. When comparing two or more groups, but with two variables (i.e. time and inhibitor/genotype) a 2-way ANOVA or a mixed effects analysis was used. To correct p-values for multiple comparisons we used the False Discovery Rate (FDR) method with the two-stage step-up to generate q-values57,58. q<0.05 was used as a cut-off to determine statistical significance. In cases where there was no multiple comparison correction (Mann–Whitney tests), p<0.05 was used as a cut-off to determine statistical significance. All statistical analysis was performed using Prism (GraphPad). p- or q-values are quoted as ranges as indicated in the Figure legends: *0.01 < p or q < 0.05, **0.001 < p or q < 0.01, ***0.0001 < p or q < 0.001, **** p or q < 0.0001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "These investigators previously demonstrated that the migration of T cells depends on the\nWNK1 kinase, its downstream kinases STK39 and OXSR1 (also called SPAK and OSR1),\nand the ion transporter regulated by SPAK and OSR1, the sodium-potassium-2-chloride\ncotransporter SLC12A2 (also called NKCC1). Here, they follow up on those studies to\nunderstand the mechanisms by which this kinase/ion transporter pathway may be controlling\ncell migration. They demonstrate decreased T cell migration in response to CCL21 in ex vivo\nassays when the activity of WNK1, SPAK/OSR1, or NKCC1, as well as the water channel\nAQP3, are decreased via genetic or pharmacological manipulations. The activated\n(phosphorylated) forms of WNK1, OSR1, and NKCC1, as well as total OSR1 and AQP3,\nare preferentially found at the leading edge of migrating T cells, where they colocalize with\nCDC42. They hypothesize that ion and water influx through NKCC1 and AQP3, regulated by\nthe WNK-SPAK/OSR1 kinase cascade, increases local ion and water influx to generate\nspace between F-actin and the plasma membrane, in order to add actin to the leading edge\nF-actin and allow migration via the Brownian ratchet mechanism. This is examined using cell\nbiological and physiological approaches.\nOverall, this is an attractive hypothesis that may be relevant not only to T cell migration, but\nalso to other cell types. Additional experiments could further bolster the findings. For\nexample, all the data on NKCC1 and AQP3 rely on a single pharmacological inhibitor for\neach transporter/channel. The Scl12a2 knockout mice are viable, and in fact, the authors\nused T cells from Slc12a2 knockouts in their previous paper, and saw effects similar to\nWNK1 knockdown or SPAK/OSR1 double knockdown, unlike in this paper in which they\nconsistently see partial effects of bumetanide. Although they argue that this could be due to\nredundant effects of four other SLC12A transporters, no information is provided about which\nof these are expressed in T cells, nor is any attempt made to experimentally test this. This is\nimportant since their model is that water moving through AQP3 follows ion influx through ion\ntransporters. Another approach to testing the role of NKCC1 would be to omit extracellular\nchloride. Genetic manipulation of AQP3 would also strengthen the findings using the AQP3\ninhibitor, eg using T cells harvested from Aqp3 knockout mice (which are viable). Another\nquestion is whether the WNK1-SPAK/OSR1 kinase cascade is regulating AQP3, since\nAQP3 inhibition fully recapitulates the phenotype observed with loss of WNK1 or\nSPAK/OSR1. This could be examined in heterologous cells.\nThe authors demonstrate that mild hypotonicity rescues the migration defect in T cells\ntreated with a very low concentration (0.2 uM) of WNK inhibitor (Fig 5C). They get a smaller\ndegree of rescue in isotonic medium with low NaCl (Fig 5D). A known consequence of\nhypotonic exposure is a decrease in intracellular chloride due to the regulatory volume\ndecrease response. Since chloride is a negative regulator of WNKs, and a very low\nconcentration of WNK inhibitor is being used in this experiment, one possibility is that the\nhypotonic medium is allowing increased activation of the residual WNK, rather than the\nwater influx per se explaining the rescue. Thus, converse to the third set of bars in this\nfigure, it would be important to lower osmolality while keeping chloride the same (or even\nhigh) to rule out this possibility. This could be done by using L-glucose in the isotonic\nmedium, and omitting it for the hypotonic. Simply lowering extracellular chloride without\nchanging tonicity may not be sufficient to lower intracellular chloride, depending on the\nchloride conductance of these cells, so the experiment performed (ie isotonic low NaCl) does\nnot rule out a possible intracellular chloride effect. Examining the degree of WNK1 S382\nphosphorylation (reflective of WNK autophosphorylation and activation) under the various\nconditions could also lend insight into this question.\nMinor:\n1. It’s a bit difficult to follow the combination of genetics and bone marrow chimeras by\nreading the Methods/Results/Figure legends. It would be helpful to have a supplemental\ntable in which this is detailed for the figures using these, ie, genotype, when tamoxifen is\napplied, which bone marrow cells are transferred into which mice, and where the T cells are\nharvested from, as well as abbreviations used (eg, “RCE” = ROSA26 CreERT2). Similarly,\n“CTV” appears in the video/figure legends without being spelled out; readers should not\nhave to go to the Methods to figure out what this non-standard abbreviation stands for. It\nwould be helpful to indicate in Figure 1C that “WNKi” = “WNK inhibitor (WNK463)”, and in 1D\nthat “SLC12A2i” = “NKCC1 inhibitor (bumetanide)” and “AQP3i” = “AQP3 inhibitor\n(DFP00173).”\n2. The authors note in the discussion that SLC12A2 inhibition reduces migration of\nglioblastoma cells. In fact, WNK1 or OSR1 knockdown also inhibits glioblastoma cell\nmigration (Zhu, Molecular Cancer 2014", "reviewer_types": "reviewer_2\": \"The authors investigate mechanisms by which WNK1 kinase and the cascade it regulates\nrelated to SLCL12 and water entry through AQP3 creates space for F-actin polymerization at\nthe leading edge of migrating T cells. 3D Motility of tumour cell by blebbing is accepted to\nrequire contraction to drive cytoplasm into blebs ahead of F-actin polymerization. There is\nalso a hydraulic engine model for motility that proposes forward movement by osmosis that\nis secondarily supported by F-actin. That authors propose that water entry through coupled\nion transport by SLC12A2 and water transport by AQP3 supports advancing the leading\nedge ahead of F-actin polymerisation. The authors build a case for this model though a\nseries of experiments focused on polarization of the key molecular players in the cascade,\nthe location of F-actin in relation to the plasma membrane, and manipulation of ions and\nosmotic gradients. The authors give only passing mention of alternative models that could be\nquantitatively tested against the authors preferred hypothesis.\n1. In Figure 1, how long were the different inhibitors applied. Its striking that WNKi reduced\ncell volume in a dose dependent manner similar to the effect of the KO, whereas SLC12A2i\nand AQPi didn’t alter cell volume. Were different times of exposure involved. The WNKi is\nmore potent at blocking migration. Could concentrations of the WNKi and AQPi that\ngenerate similar effects on motility be compared? This may result in a fair comparison that\nmay not support polarization of these signals in relation to the distribution of cytosol. The\nAQPi inhibit all water channels or just AQP3? If inhibiting all water channels shouldn’t the\neffect be greater?\n2. A complication with Figure 2 is that WNK1 and OXSR1 appear excluded from the nucleus,\nwhereas GFP is not, so most of the effect in C may be related to nuclear exclusion and\npolarization of cytoplasm. Can the authors use a form of GFP that is excluded from the\nnucleus as a control? Similarly, for SLC12A2, a membrane protein, GFP is not an\nappropriate control. The CAAX construct used later might be more suitable, although this is\npotentially complicated if SLC12A2 is not confined to the plasma membrane.\n3. Figure 3 interprets a separation of the average F-actin location from the\naverage\nmembrane location as evidence for a water filled gap between F-actin and the membrane.\nThere are two weaknesses of this approach. First, we know that the lymphocyte surface has\nsub-resolved membrane protrusion that contain F-actin, in addition to cortical f-actin and the\nextensive branched F-actin network in the leading edge that is expected to have a include F-\nactin mass that is well separated from the membrane. So the authors would need to\nconsider other models that can generate the same separation and somehow rule these out.\nThe genetics of the system also seems to impact the extent of dendritic nucleation of F-actin\nand this could be the major effect. The other issue is that building a reasonable model would\nrequire information on membrane topology that is not resolved. They would need a good\nway to go from the averaged data to a model of membrane topology for the lateral and\nleading-edge structures under different conditions to generate a model. This would require\nelectron tomography or at least SIM level super-resolution data, if not dSTORM or expansion\nmicroscopy. The observation that perturbations that alter motility also alter this measurement\ncould have many explanations that don’t involve a water filled space into which\nF-actin\npolymerizes.\n4. In Fig 4 the system used appears to confirm that inhibiting these pathways\n(WNK1,\nSLC12A2 and AQP3) reduce the degree of dendritic F-actin, which based on the relationship\nof the C-actin to the membrane would reduce the signal in this experiment. I don’t think this\nrelated to the creation of space, but more to the fraction of F-actin that is in range of the\nmembrane anchored F-acting ligand.\n5. In Figure 5 the authors apply the approach I suggested above and use the WNK1i at a 0.2\nµM to allow polarization with effects more similar to the SLC12A2 or AQP3 KO/inhibitor\nstudies. However, even with this approach, it is not clear that the results are as they would\npredict. Reducing tonicity would just non-specifically swell the cells through all aquaporins\nand this restored motility and makes the motility independent of weak inhibition of WNK1i.\nWhat happens if a higher concentration of WNKi or the knockout? Does the low tonicity\nrescue motility and F-actin flow? Are there situations where effects are reverses and WNKi\nstimulations mobility?\nI worry that there are other biophysical and biochemical effects that lead to collapse of\npolarity with WNK1i, knockout and its restoration by manipulation of cytoplasmic ion levels.\nCertainly, effect on the order seen with AQPi could be based on the proposed water entry\npromoting membrane protrusion, but the entire effect of WNK1 loss or inhibition seems well\nbeyond what can be accounted for by any osmotic effects. It should also not be forgotten\nthat WNK1 deficient cells become hyperadhesive, from earlier work from this lab, which also\nrequires actin-based contractility to activate LFA-1. How does this relate to this model? Can\nthe cells spread and generate F-actin based contractility in the interface? There are also\nNaCl dependent kinases in the cells that could also provide a mechanism for effects of NaCl\nentry.", "reviewer_3": "The manuscript by de Boer et al. builds on a previous study by the authors on the\nidentification via an RNAi screen of WNK1 as a negative regulator of integrin-mediated\nadhesion and a positive regulator of chemokine-driven migration in T cells (Köchl R. et al.,\nNat Immunol 2017). As it is the case in other cellular models, the authors had found that\ndownstream of the chemokine receptor CCR7, WNK1 activates the OXSR1 and STK39\nkinases and the SLC12A2 ion co-transporter. These previous data were suggestive of the\nfact that ion influx is required for T cell migration.\nIn the current study, the authors propose to investigate how the WNK1-SLS12A2 axis\nregulates T cell migration. They report that deficiency/inhibition or expression of mutant of\nWNK1, OXSR1, STK39 and SLS12A2 resulted in decreased migration of murine CD4+ T\ncells under confinement (under-agarose, 3D collagen-I matrix). This piece of data\nconsolidates data from their previous study. They then show the polarization of WNK1,\nOXSR1 and SLS12A2 at the leading edge of T cells migrating in confined in vitro settings.\nThis suggests that CCR7 signaling via WNK1, OXSR1 and STK39 leads to localized entry of\nNa+, K+ and Cl- ions via SLC12A2 at the leading edge of migrating T cells. The authors\nthen report that CCL21 induced a volume increase in WNK1-expressing T cells, whereas no such\nincrease was seen in the absence of WNK1, or if WNK1 was inhibited. This supports the\nhypothesis that WNK1 pathway activity results in water influx, which is required for volume\nregulation. As they report, this hypothesis aligns with the concept of the « osmotic engine\nmodel » (Stroka, K.M. et al. Cell 2014). Then, the authors focus on AQP3 as a candidate\naquaporin downstream the WNK1-SLS12A2 axis. In line with the hypothesis of the authors,\nAQP3 inhibition caused a significant decrease in the speed of CCL21-induced migration of T\ncells under agarose or in a collagen-I matrix. The authors then employ structured illumination\nmicroscopy and previously validated fluorescent probes to explore the impact of the WNK1-\nSLS12A2-AQP3 axis on the spacing between the leading edge plasma membrane and the\nunderlying actin cytoskeleton. Treatment of T cells with inhibitors targeting WNK,\nSLC12A2 or AQP3 resulted in an increase in the membrane-proximal actin distance at the\nleading edge, implying that this axis via polarized osmotic uptake of water might favor the\nactin polymerization process driving cell protrusion and migration. To complement these\nobservations, the authors measure retrograde actin flow to show that it is reduced in WNK1-\ndeficient T cells or T cells treated with inhibitors of WNK1, SLC12A2 or AQP3. Finally in\norder to more directly test the contribution of water influx in the observed process, they\nmodulate the tonicity of the medium to report increased migration speed in cells where\nWNK1 had been partially inhibited.\nOverall this is an interesting in vitro study addressing very basic aspects of polarized T cell\nmigration. Although the investigated pathway is not novel, the study provides some new\nexploration to connect chemokine sensing to polarized migration via a conserved pathway\ninvolving ion transport and osmotic regulation. The manuscript is well written and the data\nare presented in clearly organized figures. Some of the conclusions that the authors\nformulate are however to far stretched as they go beyond what is demonstrated through\nthe provided experimental work. As explained below, further work would be required to\nproperly address some of the key points the authors try to make in this study.\nMajor points :\n1- Possibility that influx of H2O2 rather than (or together with) H20 would explain the role of\nthe WNK1-SLC12A2-AQP3 axis in regulating leading-edge actin remodeling to drive\npolarized T cell migration.\nThe current study agrees with a previous publication reporting that AQP3-deficient T cells\nhave defective migration in response to various chemokines both in vitro and in vivo (Hara-\nChikuma, M. et al. Chemokine-dependent T cell migration requires aquaporin-3-mediated\nhydrogen peroxide uptake. J Exp Med 209, 1743-52, 2012). This previous publication\nhowever showed a major role of AQP3 for hydrogen peroxide uptake, which was found to\nactivate Cdc42 and polarized actin remodeling via WASP and ARP2/3. Since the authors\ndefend here an alternative mechanism (water influx facilitating actin remodeling by creating\nspace), it is essential that they decipher the relative contribution of water versus H2O2\ndriven mechanisms underlying the phenotype of AQP3-deficient or AQP3-inhibited T cells.\nThe authors would need to inhibit H202 transport to study the contribution of water influx to\nthe phenotype (migration and leading edge actin remodeling). It is also expected that the\nauthors introduce what the study by Hara-Chikuma, M. et al. showed and discuss the\nrelative H2O/H2O2 contribution.\n2- Measuring ion transport and water influx\nWhile the reported experiments (deletion or inhibition of SLC12A2 and AQP3, use of\nhypotonic medium) are suggestive of a contribution of ion transport and water influx in\npromoting actin remodeling of T cells stimulated with CCL21, direct measurements of such\nfluxes are missing. Such measurements would be quite important to sustain the claims made\nby authors in their manuscript.\nIn relation to this comment, the following sentences need to be revised according to what is\nprecisely demonstrated experimentally:\n“chemokine receptor-induced activation of the WNK1 pathway leads to water influx, most\nlikely via AQP3, which is required for actin polymerization and migration”\n“this chemokine receptor-induced water entry generates increased spacing between the\nplasma membrane and the underlying actin cytoskeleton at the leading edge”\n“WNK1 activity and ion and water influx are required to maintain reduced MPA at the leading\nedge of T cells”\n“Thus, WNK1 and ion and water influx are required for actin retrograde flow”\n3- Resolution of the F-actin-membrane distance measurements\nThe authors claim they want to investigate the fluctuating gap separating the membrane and\nthe actin filaments, which is a 10nm range phenomenon, with structured illumination\nmicroscopy. However, such approach is not expected to provide a sufficient resolution. The\nplasma membrane to “peak” F-actin signal that the authors measure seem to correspond to\nthe distance between the plasma membrane and an inner part of the lamellipodial protrusion\n(for leading edge measurement) or to the core of the cortical actin (for the cell side\nmeasurements). While the reported measurements seem to indicate an effect the tested\ninhibitions, they might only reflect an overall change in actin cytoskeleton organization.\nDifferently, the MPAct/CaaX ratio measurements appear to be a more adapted method to\nmeasure of the distribution of membrane-proximal actin. I would therefore advise to focus on\nthis latter approach.\nIn addition to the resolution issue related to the experiments conducted with SIM, the images\npresented in Figure 3 do not display the expected distribution of F-actin at the polarized T\ncell leading edge, nor the expected organization of cortical actin on the cell periphery. The\nstaining appears to be too weak and very patchy. The penetrations of LifeAct staining across\nthe membrane-orange staining are also unexpected. It would be important for the authors to\nvalidate the quality of the stainings, e.g. by costaining fixed samples with Phalloidin.\n4- Impact of the WNK1-SLC12A2-AQP3 axis on directional migration along chemokine\ngradients ?\nWhile the provided experiments provide solid evidence for the role of the WNK1-SLC12A2-\nAQP3 axis in CCL21-evoked T cell migration, they do not directly address directional\nmigration along chemokine gradients. It is felt that cell tracks, cell shape and actin\nmeasurements in the context of chemokine gradients would reinforce the current study and\nwould help understanding the precise contribution of this axis to chemokine sensing versus\ncell polarization version orientation along gradients.\n5- Which T cell motility steps are primary governed by the WNK1-SLC12A2-AQP3 axis ?\nThe authors had previously reported that WNK1 is activated downstream of CCR7, LFA-1\nand the TCR (Köchl R. et al., Nat Immunol 2017). A question of central physiological\nrelevance is therefore whether the WNK1-SLC12A2-AQP3 axis controls to a similar degree\nsteps of T cell migration known to be distinctly balanced by chemokine receptors, integrins\nand the TCR, namely trans-endothelial migration, interstitial migration and antigen-\npresenting cell scanning. Complementary in vivo and in vitro experiments would help\naddressing this point. In particular, it is well accepted that CCR7 and the TCR play rather\nopposite roles in controlling T cell motility (polarized migration versus stop of migration). The\nauthors could try unraveling how the addition of a TCR trigger to that of CCR7 alters the\ndegree of WNK1 activation and possibly the downstream axis.\n6- WNK1-SLC12A2-AQP3 axis in T cell subsets ?\nAnother direction that could greatly raise the interest for a larger community of\nimmunologists would be to assess protein expression related to the explored\npathway\n(WNK1, OXSR1, STK39, SLC12A2, AQP3) in various T cell subsets and under distinct\nactivation/differentiation conditions. Indeed various T cell subsets (Th1 vs Th2, effector T\ncells vs T regulatory cells, resting versus activated T cells) display distinct motility properties.\nWhile there has been an obvious focus on trying to relate this to the expression of specific\nchemokine receptors and adhesion receptors, much less is known on the possible tuning\nof the conserved intracellular pathways that govern motility, such as the one studied here.\nTherefore protein expression study completed with a few key functional assays with drugs\n(as done on naïve CD4+ T cells in the manuscript) might reveal very interesting aspects of\nmotility regulation underlying the properties of T cell subsets.\nFurther points requiring clarification (from figures):\nFigure 1H : increase in cell vol upon CCL21 is very limited. It is difficult if not impossible to\nassess effects (or lack of effect) of inhibitors under such settings.\nFigure 2B,C", "reviewer_e": "indicate nb of cells (to make it clearer that this comes from cell averaging).\nWhy are C and E represented differently (where is dispersion of individual cells)?\nFigure 3 : normalization of intensity values: in B both membrane and actin seem to be\nnormalized with max value = 1. In C only membrane seems to be normalized.\nFigure 4: There is experimental variability of MP Act/Caax profiles along the cell length for\nvehicule when one compares C and D panels. This variability seems to be as important as\nthe variations noted between the test conditions (WNKi, SLC12A2i, AQP3i). Please provide\nfurther evidence for the robustness of this assay and for the solidity of the data.", "reviewer_author_response": "RESPONSE TO REVIEWERS' COMMENTS\nA detailed response to the Reviewers' comments follows below, but for clarity, the\nfollowing 25 Figure panels contain new data that has been added to this revised\nversion of the manuscript:\nFigure 1D-H\nFigure 2E-I\nFigure 3D-F\nFigure 4A-B\nFigure 6C, E\nFigure S1\nFigure S2E-J\nFigure S4A-B\nThe most important new data that has been added:\n Decreased migration in cells genetically deficient for SLC12A2 or treated with\ninhibitors to SLC12A2, SLC12A6 andSLC12A7 simultaneously.\n Decreased migration of cells in medium with reduced Na+ and/or Cl-\nconcentrations.\n Direct measurement of CCL21-induced ion entry, and its dependence on\nWNK1.\n Direct measurement of CCL21-induced water entry, and its dependence on\nWNK1.\n Demonstration of increased diffusion speed of macromolecules at the front of\nthe migrating cell compared to the back, consistent with localized water entry\nat the front of the cell, and regulation of these rates by WNK1 and water influx.\nPlease note that due to the addition of the new data, Figure numbering has changed\nfrom the original version of the manuscript.\nReviewer #1 (Remarks to the Author):\nThese investigators previously demonstrated that the migration of T cells\ndepends on the WNK1 kinase, its downstream kinases STK39 and OXSR1 (also\ncalled SPAK and OSR1), and the ion transporter regulated by SPAK and OSR1,\nthe sodium-potassium-2-chloride cotransporter SLC12A2 (also called NKCC1).\nHere, they follow up on those studies to understand the mechanisms by which\nthis kinase/ion transporter pathway may be controlling cell migration. They\ndemonstrate decreased T cell migration in response to CCL21 in ex vivo assays\nwhen the activity of WNK1, SPAK/OSR1, or NKCC1, as well as the water channel\nAQP3, are decreased via genetic or pharmacological manipulations. The\nactivated (phosphorylated) forms of WNK1, OSR1, and NKCC1, as well as total\nOSR1 and AQP3, are preferentially found at the leading edge of migrating T cells,\nwhere they colocalize with CDC42. They hypothesize that ion and water influx\nthrough NKCC1 and AQP3, regulated by the WNK-SPAK/OSR1 kinase cascade,\nincreases local ion and water influx to generate space between F-actin and the\nplasma membrane, in order to add actin to the leading edge F-actin and allow\nmigration via the Brownian ratchet mechanism. This is examined using cell\nbiological and physiological approaches.\nOverall, this is an attractive hypothesis that may be relevant not only to T cell\nmigration, but also to other cell types. Additional experiments could further\nbolster the findings. For example, all the data on NKCC1 and AQP3 rely on a\n1\nsingle pharmacological inhibitor for each transporter/channel. The Scl12a2\nknockout mice are viable, and in fact, the authors used T cells from Slc12a2\nknockouts in their previous paper, and saw effects similar to WNK1 knockdown\nor SPAK/OSR1 double knockdown, unlike in this paper in which they\nconsistently see partial effects of bumetanide. Although they argue that this\ncould be due to redundant effects of four other SLC12A transporters, no\ninformation is provided about which of these are expressed in T cells, nor is any\nattempt made to experimentally test this. This is important since their model is\nthat water moving through AQP3 follows ion influx through ion transporters.\nThe reviewer is referring to Fig 7a in the Köchl et al (2016) paper where we used RNAi\nin Jurkat T cells to carry out a double knockdown of OXSR1 and STK39 or of SLC12A2\nand showed decreased CXCL12-induced migration in a Transwell assay, which was\nsimilar to a knockdown of WNK1. In the studies in this manuscript, we are working with\nprimary mouse CD4+ T cells and measuring the speed of migration in response to\nCCL21 under agarose, so it's a different system and may not be surprising that the\nresults are quantitatively different. We find that a double mutation in Oxsr1 and Stk39\nand the inhibition of SLC12A2 give less severe defects than complete loss or inhibition\nof WNK1. Nonetheless, we agree that it would be important to broaden our analysis of\nthe SLC12A-family of ion co-transporters.\nWe have added data on the expression of the SLC12A-family members (new Figure\nS2D). To complement our previous results showing that an SLC12A2 inhibitor causes\nreduced migration speed, we now show that genetic ablation of SLC12A2 gives a\nsimilar small reduction in migration speed (new Figure 1D). In addition, we have added\ndata on migration speed in cells treated with an inhibitor of SLC12A6 and SLC12A7\nalone and in combination with an SLC12A2 inhibitor (new Figure 1E). These results\nshow an additive effect of using both inhibitors together, supporting the hypothesis that\nthere is redundancy in function between SLC12A-family members.\nAnother approach to testing the role of NKCC1 would be to omit extracellular\nchloride.\nAs requested, we have measured CCL21-induced migration speed in cells where we\nreduced the concentration of NaCl thereby removing both Na+ and Cl- ions and saw a\nprogressive reduction in speed (new Figure 1G). In addition, we placed cells in medium\nwhere just Cl- ions were removed by replacing NaCl with Na gluconate and again saw\nreduced migration speed (new Figure 1H). These results support our hypothesis that\nNa+ and Cl- ions are important for chemokine-induced T cell migration.\nGenetic manipulation of AQP3 would also strengthen the findings using the\nAQP3 inhibitor, eg using T cells harvested from Aqp3 knockout mice (which are\nviable).\nSome time ago, based on published papers, we had tried to obtain AQP3-deficient\nmice, but the labs we approached were unable to provide them. More recently we have\nidentified that there is a EUCOMM allele of Aqp3 available which can be used to\ngenerate Aqp3 knockout mice. However, the process of importing and establishing this\nstrain and crossing it to a germline Cre driver to generate the knockout mice will take\na substantial amount of time and is beyond the scope of what we could practically do\nfor this response. To circumvent this, we attempted to knockout Aqp3 in primary mouse\nCD4+ T cells using CRISPR gene editing. However, despite many attempts, the best\nwe could achieve was around 30% mutation frequency, which is not good enough to\ncarry out informative migration experiments. Nonetheless, we note that AQP3-deficient\nT cells have already been shown to have defective chemokine-dependent actin\n2\npolymerization defects, as well as decreased migration in Transwells in response to\nchemokine (Hara-Chikuma et al, 2012) and we cite this paper in our manuscript.\nAnother question is whether the WNK1-SPAK/OSR1 kinase cascade is\nregulating AQP3, since AQP3 inhibition fully recapitulates the phenotype\nobserved with loss of WNK1 or SPAK/OSR1. This could be examined in\nheterologous cells.\nThe WNK1 pathway regulates AQP3 polarization. We have shown that in polarized\nmigrating T cells, AQP3 is localized to the front of the cell (Figure 3G). When WNK1 is\ninhibited or inactivated, T cells no longer polarize and there is no polarization of AQP3\n(see Figure R1). The same is true for all the other proteins analyzed in Figure 3G. We\nhave not included this data in the paper, but could do so if the reviewer wishes.\nFigure R1. AQP3 is polarized in migrating T cells\nMouse naïve CD4 T cells migrating in response to CCL21, with or without WNK463 in ICAM-1 coated\ndishes, were fixed, stained for the indicated proteins, and imaged by confocal microscopy. a, Example\nimages of cells stained for pOXSR1 demonstrating how the polarisation measure is established. White\narrow indicates starting point of line around the circumference of the cell. Scale bar, 5 µm. b, Intensity\nprofile of the circumference measurement in a. To calculate the polarisation ratio, the sum of 25% of\nthe fluorescence intensity (f.i.) signal (grey dashed lines) around the peak intensity point (red dashed\nline) is divided by the sum of the f.i. of the entire circumference. c, Graph showing the polarisation\nratio for AQP3. Each dot represents the polarisation ratio of a single cell; lines represent mean. ****q\n< 0.0001. n.s., not significant.\nConceivably, AQP3 could be regulated by changes in intracellular trafficking, and this\nmight be altered in cells with defects in the WNK1 pathway. This is difficult to study in\nprimary T cells which are very small and possess very little cytoplasm outside the\nnucleus. As the reviewer suggests this is an area of research that could be pursued in\nother cell types. However, although it is an interesting question, we feel these further\nstudies lie outside the scope of the current manuscript.\nThe authors demonstrate that mild hypotonicity rescues the migration defect in\nT cells treated with a very low concentration (0.2 uM) of WNK inhibitor (Fig 5C).\nThey get a smaller degree of rescue in isotonic medium with low NaCl (Fig 5D).\nA known consequence of hypotonic exposure is a decrease in intracellular\nchloride due to the regulatory volume decrease response. Since chloride is a\nnegative regulator of WNKs, and a very low concentration of WNK inhibitor is\nbeing used in this experiment, one possibility is that the hypotonic medium is\nallowing increased activation of the residual WNK, rather than the water influx\n3\nper se explaining the rescue. Thus, converse to the third set of bars in this\nfigure, it would be important to lower osmolality while keeping chloride the same (or\neven high) to rule out this possibility. This could be done by using L-glucose in\nthe isotonic medium, and omitting it for the hypotonic. Simply lowering\nextracellular chloride without changing tonicity may not be sufficient to lower\nintracellular chloride, depending on the chloride conductance of these cells, so\nthe experiment performed (ie isotonic low NaCl) does not rule out a possible\nintracellular chloride effect. Examining the degree of WNK1 S382\nphosphorylation (reflective of WNK autophosphorylation and activation) under\nthe various conditions could also lend insight into this question.\nThe reviewer suggests that the 20% reduction in the concentration of chloride in the\nhypotonic medium in Figure 7C, 7D may be activating WNK1, despite the presence of\na low dose of WNK inhibitor, and that this accounts for the rescue in migration speed.\nTo test this idea, we needed to analyze WNK1 activity, which we have been doing by\nmeasuring levels of p-OXSR1 (see for example Figure S2C). Unfortunately, the current\nbatch of polyclonal, sheep serum-derived anti-p-OXSR1 antibodies does not give\nreliable detection of the p-OXSR1 protein on immunoblots, and despite numerous\nattempts, we have not been able to address this point. We have edited the text to refer\nto the possibility that low chloride may be activating WNK1 (page 16, paragraph 1).\nMinor:\n1. It’s a bit difficult to follow the combination of genetics and bone marrow\nchimeras by reading the Methods/Results/Figure legends. It would be helpful to\nhave a supplemental table in which this is detailed for the figures using these,\nie, genotype, when tamoxifen is applied, which bone marrow cells are\ntransferred into which mice, and where the T cells are harvested from, as well as\nabbreviations used (eg, “RCE” = ROSA26 CreERT2). Similarly, “CTV” appears in\nthe video/figure legends without being spelled out; readers should not have to\ngo to the Methods to figure out what this non-standard abbreviation stands for.\nIt would be helpful to indicate in Figure 1C that “WNKi” = “WNK inhibitor\n(WNK463)”, and in 1D that “SLC12A2i” = “NKCC1 inhibitor (bumetanide)” and\n“AQP3i” = “AQP3 inhibitor (DFP00173).”\nTo address the reviewer's point, we have generated new Figures showing how CD4+\nT cells deficient in either WNK1 or OXSR1 and STK39 were made following generation\nof bone marrow chimeras and subsequent tamoxifen treatment (new Figure S1A-B).\nWithin the legends of these, RCE is defined. In addition, we have explained the\nprocedure by which we generated WNK1-deficient T cells in the Results (page 5,\nparagraph 2). As requested, CTV and all inhibitors are defined in the legends of all\nFigures they appear in.\n2. The authors note in the discussion that SLC12A2 inhibition reduces migration\nof glioblastoma cells. In fact, WNK1 or OSR1 knockdown also inhibits\nglioblastoma cell migration (Zhu, Molecular Cancer 2014", "reviewer_": "n4\nThe authors investigate mechanisms by which WNK1 kinase and the cascade it\nregulates related to SLCL12 and water entry through AQP3 creates space for F-\nactin polymerization at the leading edge of migrating T cells. 3D Motility of\ntourour cell by blebbing is accepted to require contraction to drive cytoplasm\ninto blebs ahead of F-actin polymerization. There is also a hydraulic engine\nmodel for motility that proposes forward movement by osmosis that is\nsecondarily supported by F-actin. That authors propose that water entry through\ncoupled ion transport by SLC12A2 and water transport by AQP3 supports\nadvancing the leading edge ahead of F-actin polymerisation. The authors build\na case for this model though a series of experiments focused on polarization of\nthe key molecular players in the cascade, the location of F-actin in relation to\nthe plasma membrane, and manipulation of ions and osmotic gradients. The\nauthors give only passing mention of alternative models that could be\nquantitatively tested against the authors preferred hypothesis.\n1. In Figure 1, how long were the different inhibitors applied. Its striking that\nWNKi reduced cell volume in a dose dependent manner similar to the effect of\nthe KO, whereas SLC12A2i and AQPi didn’t alter cell volume. Were different\ntimes of exposure involved.\nFor the migration assays under agarose shown in Figure 1, cells were injected under\nagarose containing the inhibitors at least 30 min before imaging was started. For the\ncell volume measurements shown in Figure 2A-B, cells were treated with inhibitors for\n20 min, with CCL21 (or vehicle) for a further 20 min and then volume was measured.\nDetails of timings are listed in the Methods. For each protocol, cells were exposed to\nall inhibitors for the same amount of time.\nThe WNKi is more potent at blocking migration. Could concentrations of the\nWNKi and AQPi that generate similar effects on motility be compared? This may\nresult in a fair comparison that may not support polarization of these signals in\nrelation to the distribution of cytosol.\nAs suggested by the reviewer, we have titered the dose of WNKi, examining the effect\non migration speed and circularity (a measure of polarization) (Figure S2D). We found\nthat 5 µM WNKi resulted in a maximal inhibition of CCL21-induced migration and\npolarization, 0.5 µM WNKi partially inhibited migration and polarization, whereas 0.2\nµM WNK1 reduced migration speed by a small amount, but critically, had no effect on\npolarization. In many assays we have used these sub-maximal (0.2 and 0.5 µM) doses\nof WNKi in order to allow us to examine the roles of WNK1 in cells that still polarized,\nmaking a fairer comparison with inhibition of SLC12A2 or AQP3 (Figures 1C, 2B, 2F,\n4B, 6C, 6E, 7B, 7C-E, S2F, S3C). We have also added data on the effects of WNK1\ndeficiency as well as the effects of the inhibitors on circularity (new Figures S2E, F, H).\nThe AQPi inhibit all water channels or just AQP3? If inhibiting all water channels\nshouldn’t the effect be greater?\nRNA-seq data shows that mouse CD4+ T cells express AQP3, AQP9 and AQP11 (new\nFigure 2H). DFP00173 inhibits AQP3, but not AQP7 or AQP9 (Sonntag et al, 2019);\nwe are not aware if DFP00173 inhibits AQP11. Thus, treatment with DFP00173 inhibits\nAQP3 but leaves AQP9 and maybe AQP11 active. This information has been added\nto the text.\n2. A complication with Figure 2 is that WNK1 and OXSR1 appear excluded from\nthe nucleus, whereas GFP is not, so most of the effect in C may be related to\n5\nnuclear exclusion and polarization of cytoplasm. Can the authors use a form of\nGFP that is excluded from the nucleus as a control?\nTo address the reviewer's point, we imaged a nuclear-excluded GFP and show that\nthis does not accumulate at the leading edge (new Figure 3D-F). Thus, the\naccumulation of GFP-WNK1, GFP-OXSR1 and GFP-SLC12A2 at the leading edge is\nnot due to nuclear exclusion of these fusion proteins. It is also not a function of passive\nenrichment of WNK1 pathway proteins at the leading edge, as the nucleus-excluded\nGFP localizes to the rear of the cell.\nSimilarly, for SLC12A2, a membrane protein, GFP is not an appropriate control.\nThe CAAX construct used later might be more suitable, although this is\npotentially complicated if SLC12A2 is not confined to the plasma membrane.\nWe confirmed the localization of SLC12A2 and p-SLC12A2 at the leading edge in\nFigure 3G, H where we image fixed cells and compare to the localization of CDC42\nand CD44 that mark the membrane at the leading and trailing edges respectively. In\naddition, we note that the plasma membrane-tethered CAAX construct referred to by\nthe reviewer, is uniformly distributed around the migrating cell, with no preferential\naccumulation at the leading edge (Figures 6A, B).\n3. Figure 3 interprets a separation of the average F-actin location from the\naverage membrane location as evidence for a water filled gap between F-actin\nand the membrane.\nThere are two weaknesses of this approach. First, we know that the lymphocyte surface has\nsub-resolved membrane protrusion that contain F-actin, in addition to cortical f-actin and the\nextensive branched F-actin network in the leading edge that is expected to have a include F-\nactin mass that is well separated from the membrane. So the authors would need to\nconsider other models that can generate the same separation and somehow rule these out.\nThe genetics of the system also seems to impact the extent of dendritic nucleation of F-actin\nand this could be the major effect. The other issue is that building a reasonable model would\nrequire information on membrane topology that is not resolved. They would need a good\nway to go from the averaged data to a model of membrane topology for the lateral and\nleading-edge structures under different conditions to generate a model. This would require\nelectron tomography or at least SIM level super-resolution data, if not dSTORM or expansion\nmicroscopy. The observation that perturbations that alter motility also alter this measurement\ncould have many explanations that don’t involve a water filled space into which\nF-actin\npolymerizes.\nWe agree with the reviewer that the observation of an increased distance between the\npeak of cortical F-actin density and the plasma membrane as measured by iSIM (now\nFigure 5) does not unequivocally show that there is increased spacing between the\nmembrane and the underlying F-actin. There could, for example, be a wider band of\nF-actin at the leading edge in cells that are migrating faster, and inhibitors that slow\nthe cells down, may also cause a less wide band of F-actin. We have amended the\ntext to reflect this.\nThe suggestion to image the F-actin at the leading edge by EM tomography is a good\none. We spent several months in collaboration with experts trying this approach as\nwell as STORM imaging, but we were unable to resolve the actin structure in the\nleading edge of migrating T cells. This is probably because the protruding leading edge\nis small and the actin cytoskeleton is too dense. Thus, unfortunately, we feel these\nexperiments are beyond the scope of this manuscript.\n6\n4. In Fig 4 the system used appears to confirm that inhibiting these pathways\n(WNK1, SLC12A2 and AQP3) reduce the degree of dendritic F-actin, which based\non the relationship of the C-actin to the membrane would reduce the signal in\nthis experiment. I don’t think this related to the creation of space, but more to\nthe fraction of F-actin that is in range of the membrane anchored F-acting ligand.\nWe agree with the reviewer that the MPAct probe reports on the amount of F-actin\nwithin 10 nm of the membrane rather than directly reporting on the space (now in\nFigure 6).\nWork from the Sixt lab (Mueller et al, 2017) shows that in migrating cells, a reduced\nload of the membrane on actin filaments at the leading edge results in rapid\npolymerization of actin filaments perpendicular to the membrane. These outcompete\na network of filaments at shallow angles, thereby generating a region with lower F-\nactin density proximal to the plasma membrane. We hypothesize that water entry at\nthe front of the cell would contribute to this reduced load and hence to lower membrane\nproximal F-actin (MPA) at the leading edge.\nOur data in Figure 6 test this hypothesis. These show that there is less MPA at the\nleading edge than at the trailing edge, and that inhibition of WNK1, SLC12A2 or AQP3\ncauses an increase in the amount of MPA at the leading edge, consistent with this\nhypothesis. We have ensured that the manuscript text does not state that the MPAct\nprobe directly reports on space between the membrane and the actin cytoskeleton.\n5. In Figure 5 the authors apply the approach I suggested above and use the\nWNK1i at a 0.2 µM to allow polarization with effects more similar to the SLC12A2\nor AQP3 KO/inhibitor studies. However, even with this approach, it is not clear\nthat the results are as they would predict. Reducing tonicity would just non-\nspecifically swell the cells through all aquaporins and this restored motility and\nmakes the motility independent of weak inhibition of WNK1i.\nAs the reviewer suggested earlier, we used the low dose of WNKi (0.2 µM) because\nthis partially inhibits migration but does not affect polarization of the cell, and more\nclosely resembles the effect of inhibition of SLC12A2 or AQP3. Under these conditions,\nhypotonic medium rescues the migration defect (now Figure 7C-D). It also rescues\nactin flow rates (Figure 7E) and restores reduced MPA at the leading edge (Figure 6E).\nThe reviewer points out that the hypotonic treatment may cause water entry all around\nthe cell and not just at the leading edge. This may be the case, though we note that\nAQP3 is polarized to the leading edge, so more water may enter at the leading edge\nduring hypotonic treatment. In any case, we propose that it is only at the leading edge\nthat water entry would be able to promote migration, since this is where the motility\nmachinery of actin polymerization is polarized in response to chemokine receptor\nsignaling. Our hypothesis is that the water entry would facilitate actin polymerization at\nthe leading edge, not that it would stimulate actin polymerization all around the cell.\nTo further support our hypothesis that WNK1 activation at the leading edge causes\nlocalized water entry we transfected T cells with Qdots, brightly fluorescent\nnanocrystals, and measured their diffusion rates. We found that Qdots diffused faster\nat the front of the cell compared to the back, consistent with localized water entry at\nthe front leading to a less dense cytoplasm (new Figure 4). Furthermore, the diffusion\nof Qdots was slowed down by partial inhibition of WNKi, and this was rescued by\nputting the cells into hypotonic medium. Importantly, Qdot diffusion speeds were faster\nat the leading edge than in the rear of the cell even in the hypotonic-rescued cells,\n7\nconsistent with continued polarization of water influx even in the hypotonic rescue of\nlow-dose WNKi-treated cells.\nWhat happens if a higher concentration of WNKi or the knockout? Does the low\ntonicity rescue motility and F-actin flow?\nAt high doses (e.g. 5 µM WNKi) the cells no longer polarize or migrate and hypotonic\nmedium does not rescue migration. We speculate that WNK1 may be playing other\nroles in the cell, including contributing to the formation of a polarized morphology.\nAre there situations where effects are reverses and WNKi stimulations mobility?\nWe have never seen WNK1 inhibition increasing motility at any dose (see Figure S2D).\nI worry that there are other biophysical and biochemical effects that lead to\ncollapse of polarity with WNK1i, knockout and its restoration by manipulation of\ncytoplasmic ion levels.\nCertainly, effect on the order seen with AQPi could be based on the proposed water entry\npromoting membrane protrusion, but the entire effect of WNK1 loss or inhibition seems well\nbeyond what can be accounted for by any osmotic effects.\nWe agree with the reviewer. At a high dose of WNK inhibitor, or in WNK1-deficient\ncells there is a complete loss of polarization of the cells and this cannot be reversed\nwith hypotonic medium. We agree that WNK1 must additionally be doing something\nother than regulating water entry at the leading edge. We have amended the\nDiscussion to make this point clear.\nIt should also not be forgotten that WNK1 deficient cells become hyperadhesive,\nfrom earlier work from this lab, which also requires actin-based contractility to\nactivate LFA-1. How does this relate to this model? Can the cells spread and\ngenerate F-actin based contractility in the interface? There are also\nNaCl dependent kinases in the cells that could also provide a mechanism for effects\nof NaCl entry.\nIn the Köchl et al (2016) paper we investigated the relationship between the increased\nadhesion and reduced migration of WNK1-deficient T cells. It was possible that the\nincreased adhesion could be responsible for the reduced rate of migration. However,\nwe were able to show that this was not true. If we blocked integrin adhesion, the cells\nslowed down even further (Fig 4c of Köchl et al), demonstrating that the adhesive and\nmigratory phenotypes are distinct. Furthermore, in this earlier publication we showed\nknockout or knockdown of SLC12A2 reduced migration but had no effect on adhesion\n(Fig 6a, b, d of Köchl et al), again supporting the view that the adhesive and migratory\nphenotypes of WNK1-deficient T cells are distinct phenotypes. This point is discussed\nin the Introduction of the current manuscript (page 4, paragraph 2).\nReviewer #3 (Remarks to the Author):\nThe manuscript by de Boer et al. builds on a previous study by the authors on\nthe identification via an RNAi screen of WNK1 as a negative regulator of integrin-\nmediated adhesion and a positive regulator of chemokine-driven migration in T\ncells (Köchl R. et al., Nat Immunol 2017). As it is the case in other cellular models,\nthe authors had found that downstream of the chemokine receptor CCR7, WNK1\nactivates the OXSR1 and STK39 kinases and the SLC12A2 ion co-transporter.\ncell migration.\nIn the current study, the authors propose to investigate how the WNK1-SLS12A2\naxis regulates T cell migration. They report that deficiency/inhibition or\nexpression of mutant of WNK1, OXSR1, STK39 and SLS12A2 resulted in\ndecreased migration of murine CD4+ T cells under confinement (under-agarose,\n3D collagen-I matrix). This piece of data consolidates data from their previous\nstudy. They then show the polarization of WNK1, OXSR1 and SLS12A2 at the\nleading edge of T cells migrating in confined in vitro settings. This suggests that\nCCR7 signaling via WNK1, OXSR1 and STK39 leads to localized entry of\nNa+, K+ and Cl- ions via SLC12A2 at the leading edge of migrating T cells. The authors\nthen report that CCL21 induced a volume increase in WNK1-expressing T cells,\nwhereas no such increase was seen in the absence of WNK1, or if WNK1 was\ninhibited. This supports the hypothesis that WNK1 pathway activity results in\nwater influx, which is required for volume regulation. As they report, this\nhypothesis aligns with the concept of the « osmotic engine\nmodel » (Stroka, K.M.\net al. Cell 2014). Then, the authors focus on AQP3 as a candidate aquaporin\ndownstream the WNK1-SLS12A2 axis. In line with the hypothesis of the authors,\nAQP3 inhibition caused a significant decrease in the speed of CCL21-induced\nmigration of T cells under agarose or in a collagen-I matrix. The authors then\nemploy structured illumination microscopy and previously validated fluorescent\nprobes to explore the impact of the WNK1-SLS12A2-AQP3 axis on the spacing\nbetween the leading edge plasma membrane and the underlying actin\ncytoskeleton. Treatment of T cells with inhibitors targeting WNK, SLC12A2 or\nAQP3 resulted in an increase in the membrane-proximal actin distance at the\nleading edge, implying that this axis via polarized osmotic uptake of water might\nfavor the actin polymerization process driving cell protrusion and migration. To\ncomplement these observations, the authors measure retrograde actin flow to\nshow that it is reduced in WNK1-deficient T cells or T cells treated with inhibitors\nof WNK1, SLC12A2 or AQP3. Finally in order to more directly test the\ncontribution of water influx in the observed process, they modulate the tonicity\nof the medium to report increased migration speed in cells where WNK1 had\nbeen partially inhibited.\nOverall this is an interesting in vitro study addressing very basic aspects of\npolarized T cell\nmigration. Although the investigated pathway is not novel, the study provides some new\nexploration to connect chemokine sensing to polarized migration via a conserved pathway\ninvolving ion transport and osmotic regulation. The manuscript is well written and the data\nare presented in clearly organized figures. Some of the conclusions that the authors\nformulate are however to far stretched as they go beyond what is demonstrated through\nthe provided experimental work. As explained below, further work would be required to\nproperly address some of the key points the authors try to make in this study.\nMajor points:\n1- Possibility that influx of H2O2 rather than (or together with) H20 would explain\nthe role of the WNK1-SLC12A2-AQP3 axis in regulating leading-edge actin\nremodeling to drive polarized T cell migration.\nThe current study agrees with a previous publication reporting that AQP3-\ndeficient T cells have defective migration in response to various chemokines\nboth in vitro and in vivo (Hara-\nChikuma, M. et al. Chemokine-dependent T cell\nmigration requires aquaporin-3-mediated hydrogen peroxide uptake. J Exp Med\n209, 1743-52, 2012). This previous publication however showed a major role of\nAQP3 for hydrogen peroxide uptake, which was found to activate Cdc42 and\npolarized actin remodeling via WASP and ARP2/3. Since the authors defend here\nan alternative mechanism (water influx facilitating actin remodeling by creating\nspace), it is essential that they decipher the relative contribution of water versus\nH2O2 driven mechanisms underlying the phenotype of AQP3-deficient or AQP3-\ninhibited T cells. The authors would need to inhibit H202 transport to study the\ncontribution of water influx to the phenotype (migration and leading edge actin\nremodeling). It is also expected that the authors introduce what the study by\nHara-Chikuma, M. et al. showed and discuss the relative H2O/H2O2 contribution.\nAs requested, we have investigated the role of H O in T cell migration, since AQP3\n2 2\ncan transport H O as well as water. Using catalase to eliminate H O , we show that\n2 2 2 2\nH O is not required for CCL21-induced migration of mouse CD4+ T cells (new Figure\n2 2\nS4B). It is not clear why our results are different from those reported in Hara-Chikuma\net al 2012. There are a number of methodological differences. We measured migration\nspeed in response to CCL21 under agarose, whereas Hara-Chikuma et al measured\n% migration through a Transwell in response to CXCL12. In addition, we used 20 U/ml\ncatalase whereas Hara-Chikuma et al used 2000 U/ml catalase. Our choice of 20 U/ml\nwas based on a titration where we investigated how much catalase is needed to\neliminate 1 mM H O as detected by DCFDA fluorescence. We found that 20 U/ml was\n2 2\nsufficient for this purpose, with no further improvement seen at 200 U/ml (new Figure\nS4A). Thus, we chose 20 U/ml catalase, since this was the lowest concentration that\nwas sufficient to eliminate 1 mM H O . It is possible that the supramaximal 2000 U/ml\n2 2\ncatalase used by Hara-Chikuma et al may have had off-target effects that\ncompromised migration. In any case, the hypotonic rescue experiments shown in\nFigure 7 demonstrate that water entry is able to reverse the migration defects in WNK1-\ninihibited cells, supporting the importance of water flux in T cell migration.\n2- Measuring ion transport and water influx\nWhile the reported experiments (deletion or inhibition of SLC12A2 and AQP3,\nuse of hypotonic medium) are suggestive of a contribution of ion transport and\nwater influx in promoting actin remodeling of T cells stimulated with CCL21,\ndirect measurements of such fluxes are missing. Such measurements would be\nquite important to sustain the claims made by authors in their manuscript.\nWe agree with the reviewer that it would be important to directly demonstrate CCL21-\ninduced ion and water flux. We used inductively-coupled plasma mass spectrometry\nto measure ion content in T cells and found the CCL21 stimulation leads to an increase\nin K+ ions in the cell which was blocked when WNK1 was inhibited (new Figure 1F).\nWe were unable to get reliable data for Na+ or Cl- because the concentration of these\nions is high in the extracellular medium resulting in a high background of these ions in\nthe samples, precluding accurate measurements of these ions.\nIn addition, we directly measured water uptake using deuterated water (2H O) and then\ninhibition (new Figure 2G), closely matching the volume changes resulting from CCL21\nstimulation and WNK1 inhibition.\nIn relation to this comment, the following sentences need to be revised\naccording to what is precisely demonstrated experimentally:\n10\n“chemokine receptor-induced activation of the WNK1 pathway leads to water\ninflux, most likely via AQP3, which is required for actin polymerization and\nmigration”\n“this chemokine receptor-induced water entry generates increased spacing\nbetween the plasma membrane and the underlying actin cytoskeleton at the\nleading edge”\n“WNK1 activity and ion and water influx are required to maintain reduced MPA\nat the leading edge of T cells”\n“Thus, WNK1 and ion and water influx are required for actin retrograde flow”\nAll of these sentences have been edited to ensure that they accurately represent our\nfindings. In addition, we have edited other parts of the text in a similar way to make\nsure that we specify our results precisely.\n3- Resolution of the F-actin-membrane distance measurements\nThe authors claim they want to investigate the fluctuating gap separating the\nmembrane and the actin filaments, which is a 10nm range phenomenon, with\nstructured illumination microscopy. However, such approach is not expected to\nprovide a sufficient resolution. The plasma membrane to “peak” F-actin signal\nthat the authors measure seem to correspond to the distance between the\nplasma membrane and an inner part of the lamellipodial protrusion\n(for leading edge measurement) or to the core of the cortical actin (for the cell side\nmeasurements). While the reported measurements seem to indicate an effect the\ntested inhibitions, they might only reflect an overall change in actin cytoskeleton\norganization. Differently, the MPAct/CaaX ratio measurements appear to be a\nmore adapted method to measure of the distribution of membrane-proximal\nactin. I would therefore advise to focus on this latter approach.\nIn addition to the resolution issue related to the experiments conducted with\nSIM, the images presented in Figure 3 do not display the expected distribution\nof F-actin at the polarized T cell leading edge, nor the expected organization of\ncortical actin on the cell periphery. The staining appears to be too weak and very\npatchy. The penetrations of LifeAct staining across the membrane-orange\nstaining are also unexpected. It would be important for the authors to validate\nthe quality of the stainings, e.g. by costaining fixed samples with Phalloidin.\nThe patchy appearance of the images, including the apparent penetration of LifeAct\nsignal through the membrane-orange staining, is due to use of a high resolution\nimaging method (iSIM) and high speed of acquisition with a 200 ms exposure every\n250 ms. Thus, these images will never look like confocal images of fixed cells stained\nwith phalloidin. The analysis of fixed cells is unlikely to help with this, since there is a\ndanger that fixation may change the morphology of the structures being imaged at sub-\nµm resolution.\n4- Impact of the WNK1-SLC12A2-AQP3 axis on directional migration along\nchemokine gradients ?\nWhile the provided experiments provide solid evidence for the role of the WNK1-SLC12A2-\nAQP3 axis in CCL21-evoked T cell migration, they do not directly\naddress directional migration along chemokine gradients. It is felt that cell\ntracks, cell shape and actin measurements in the context of chemokine\n11\ngradients would reinforce the current study and would help understanding the\nprecise contribution of this axis to chemokine sensing versus cell polarization\nversion orientation along gradients.\nAs requested, we have added analysis of the role of WNK1 in directional migration\n(chemotaxis). Using activated T cells migrating through a collagen-I matrix in response\nto a gradient of CXCL12, we were able to show that WNK1 is required for their\ndirectional migration (new Figure S2J).\n5- Which T cell motility steps are primary governed by the WNK1-SLC12A2-AQP3\naxis ?\nThe authors had previously reported that WNK1 is activated downstream of\nCCR7, LFA-1 and the TCR (Köchl R. et al., Nat Immunol 2017). A question of\ncentral physiological relevance is therefore whether the WNK1-SLC12A2-AQP3\naxis controls to a similar degree steps of T cell migration known to be distinctly\nbalanced by chemokine receptors, integrins and the TCR, namely trans-\nendothelial migration, interstitial migration and antigen-\npresenting cell scanning. Complementary in vivo and in vitro experiments would help\naddressing this point. In particular, it is well accepted that CCR7 and the TCR\nplay rather opposite roles in controlling T cell motility (polarized migration versus\nstop of migration). The authors could try unraveling how the addition of a TCR\ntrigger to that of CCR7 alters the degree of WNK1 activation and possibly the\ndownstream axis.\nMost of the questions raised by the reviewer were addressed in our earlier publication\n(Köchl et al, 2016). In this earlier study we analyzed the in vivo migration of WNK1-\ndeficient and control T cells and showed they entered lymph nodes less efficiently,\nbeing more likely to be associated with lumen and perivascular areas of the lymph\nnode, they adhered more strongly to endothelium, but migrated more slowly on it and\ntransmigrated more slowly through it (Fig 3a-g). Using intra-vital imaging, we showed\nthat they migrated more slowly in lymph nodes once they have entered them (Fig 4a-\nd). Furthermore, we showed that the decreased rate of migration of the WNK1-deficient\ncells was not due to their hyperadhesive integrins. Blocking integrin binding in vivo\ncaused the WNK1-deficient T cells to slow down even further, showing that if anything,\nthe cells were using their hyperadhesive integrins to promote their migration (Fig 4c).\nWe refer to these results in the Introduction. We feel that further studies, e.g. on the\nefficiency of scanning APCs, although interesting, is outside the scope of the current\nstudy.\n6- WNK1-SLC12A2-AQP3 axis in T cell subsets?\nAnother direction that could greatly raise the interest for a larger community of\nimmunologists would be to assess protein expression related to the explored\npathway (WNK1, OXSR1, STK39, SLC12A2, AQP3) in various T cell subsets and\nunder distinct activation/differentiation conditions. Indeed various T cell\nsubsets (Th1 vs Th2, effector T cells vs T regulatory cells, resting versus\nactivated T cells) display distinct motility properties.\nWhile there has been an obvious focus on trying to relate this to the expression of specific\nchemokine receptors and adhesion receptors, much less is known on the possible tuning\nof the conserved intracellular pathways that govern motility, such as the one\nstudied here. Therefore protein expression study completed with a few key\nfunctional assays with drugs (as done on naïve CD4+ T cells in the manuscript)\nmight reveal very interesting aspects of motility regulation underlying the\nproperties of T cell subsets.\ncells (new Figure S2I-J). We have previously shown that WNK1 is required for the\nmigration and lymph node homing of CD8+ T cells (Köchl et al 2016), for the CXCL12-\ninduced migration of CD4-CD8- thymocytes (Köchl et al 2020) and more recently for\nthe CXCL13-induced migration of B cells in vitro and in vivo (Hayward et al 2023). We\nhave cited these studies in the manuscript. Thus, a picture is emerging that this\npathway is of importance for the migration of many immune (and non-immune cell\ntypes). Further studies on the role of the WNK pathway in the migration of different T\ncell subsets (Th1, Th2, Th17 and Treg) would be an interesting area to pursue, but we\nfeel these are outside the scope of the current study.\nNonetheless, inspection of a published RNA-seq dataset on CD4+ T cell Th subsets,\nwhich includes naive, Th1, Th2, Th17, iTreg and Treg cells (Th-Express"}, "author_response": ""}]

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