Pak1 Kinase Promotes Activated T Cell Trafficking by Regulating the Expression of L-Selectin and CCR7

Abstract

Normal function of the adaptive immune system requires trafficking of T cells between the blood and lymphoid organs. Lymphocyte homing to lymph nodes requires that they cross endothelial barriers present in blood vessels and lymphatics. This multi-step process requires a remodeling of the lymphocyte plasma membrane, which is mediated by the dynamic re-arrangement of the actin cytoskeleton. Pak1 plays a central role in cell morphology, adhesion and migration in various cell types. Here we demonstrate that Pak1 is required for activated CD4⁺ T cell trafficking to lymph nodes. Pak1 deficiency in T cells causes a defect in the transcription of CCR7 and L-selectin, thereby altering lymphocyte trafficking. Additionally, we report an increase in L-selectin shedding in Pak1-deficient T cells, which correlates with a decrease in the recruitment of calmodulin to the cytoplasmic tail of L-selectin during T cell activation. Overall, our findings demonstrate that by regulating the expression of two major lymph node homing molecules, L-selectin and CCR7, Pak1 mediates activated CD4⁺ T cell trafficking.

Keywords: CCR7; L-selectin; L-selectin shedding; Pak1 kinase; lymph node trafficking.

Figure 1

Pak1 is required for efficient homing of activated CD4⁺ T cells into lymph nodes. (A) Schematic of in vitro CD4⁺ T cell activation and co-transfer 1:1 of differentially dye-labeled WT and Pak1(T)^−/− T cell blasts in recipient C57BL/6 for T cell trafficking to lymphoid tissues by flow cytometry quantification. (B) Recovery of co-transferred labeled WT or Pak1(T)^−/− cells in A, presented as a percentage of total transferred cells recovered from the blood, LNs or spleen at 1 h after transfer. Each dot indicates result from an individual mouse; horizontal bars indicate the mean. Data were pooled from four to five recipient mice of three to four independent experiments. Sample sizes: 21, blood and spleen; 19 lymph nodes. (C) 2-photon intravital image of the HEV at 15 min after transfer of WT, green; and Pak1(T)^−/−, red; CD4⁺ T cell blasts. HEV are visualized with an anti-PNAd-Pacific blue antibody (Blue). Box 1 and box 2 indicate a region with a WT and a Pak1(T)^−/− CD4⁺ T cell, respectively, prior to their extravasation. Scale bar, 40 μm. (D) Panels show zoomed-in views of WT (green cells top, Box 1) and Pak1(T)^−/− (red cells bottom, Box 2) CD4⁺ T cell blasts moving from the inside of the vessel to the outside. t₀ indicates the first time the cell adhered to the wall of the HEVs. White arrows indicate extravasating cells. White dashed lines indicate the vessel wall. Scale bars, 6 μm. (E) Left, 2-photon intravital image 2 h after WT and Pak1(T)^−/− CD4⁺ T cell blast transfer. HEV are labeled in gray. Scale bar, 40 μm. Right, automated tracking of CD4⁺ T lymphocytes migration. Trajectories of WT CD4⁺ T cell blasts are displayed as green tracks while Pak1(T)^−/− CD4⁺ T cell blasts are displayed in red. Scale bar, 50 μm. (F) Analysis of cell populations in the inguinal node at various time points from 15 to 60 min after cell transfer into the tail vein. Percentage of each population of the total is shown. Pooled from three independent experiments. (G) Inguinal lymph nodes explanted 2 h after adoptive transfer of dye-labeled WT and Pak1(T)^−/− CD4⁺ blast T cells were cleared following the tissue clearing process using FocusClear for 10 h. After clearing, lymph nodes were imaged on a 2-photon microscope. The panels show a 3D volume rendering image of individual dye-labeled WT (Green dye) and Pak1(T)^−/− (Red dye) CD4⁺ blast T cells and a merge. Right, quantification of dye-labeled WT and Pak1(T)^−/− CD4⁺ blast T cells in inguinal lymph nodes subjected to tissue clearing. Percentage of each population of the total is shown. Pooled from five LNs of three independent experiments. Scale bars, 250 μm. Statistical analysis: unpaired Student's t-test (B,F,G). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2

Pak1 regulates L-selectin and CCR7 transcription in activated CD4⁺ T cells. (A) Left, L-selectin surface marker expression as measured by flow cytometry of blast T cells. Representative histograms from eight independent experiments showing a comparison of L-selectin expression between WT and Pak1(T)^−/− are shown. Bar chart shows MFI percentage, mean fluorescence intensity, of L-selectin. Right, CCR7 surface marker expression as measured by flow cytometry in CD4⁺ T cell blasts from WT and Pak1(T)^−/− mice. The results are representative of four experiments. Bar chart shows MFI (%) of CCR7 in WT and Pak1(T)^−/− blast T cells. (B) Relative expression of Sell and Ccr7 mRNA by qRT-PCR in CD4⁺ T cell blasts from WT and Pak1(T)^−/− mice. Pooled from six independent experiments. (C) LFA-1 surface marker expression as measured by flow cytometry in CD4⁺ T cell blasts from WT and Pak1(T)^−/− mice. LFA-1 expression bar chart shows MFI (%) of LFA-1 from three experiments. (D) Quantification of the percentage of L-selectin-negative cells (L-selectin^lo) and L-selectin-positive cells (L-selectin^hi) in (A). The results are representative of six experiments. (E) Differentially dye-labeled CD4⁺ WT or Pak1(T)^−/− blast T cells were co-adaptively transferred intravenously at a 1:1 ratio, and T cell trafficking to lymph nodes was quantified by flow cytometry. Recovery of co-transferred WT or Pak1(T)^−/− blast T cells, (left) presented as a percentage of total transferred cells, or (right) presented as a percentage of L-selectin^hi expressing cells recovered from the LNs at 1 h after transfer. Each dot indicates result from an individual mouse; horizontal bars indicate the mean. Data were pooled from five recipient mice. Statistical analysis: unpaired Student's t-test (A–C,E) ANOVA between selected columns with Sidak's multiple comparison test (D). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3

Regulation of Foxo1/Klf2 pathway by Pak1/JNK. (A) Scheme of Foxo1 nuclear localization regulation. (B) Relative expression of Klf2 mRNA by qRT-PCR in CD4⁺ T cell blasts from WT and Pak1(T)^−/− mice. Data were pooled from five independent experiments. (C) Lamin-B nuclear and tubulin-enriched cytosolic fractions were prepared from WT and Pak1(T)^−/− CD4⁺ T blasts. Foxo1 nuclear translocation was studied by western-blot. Foxo1 levels in cytosol or nucleus were normalized to tubulin or lamin B, respectively. Numbers indicate the relative abundance of Foxo1 in comparison to WT cells. Results from one of more than three independent experiments are shown. (D) Lysates from WT and Pak1(T)^−/− CD4⁺ T blasts were analyzed with the indicated antibodies to measure total Foxo1; bottom, abundance of Foxo1 relative to that of β-actin pooled from five independent experiments. (E) WT and Pak1(T)^−/− CD4⁺ T blasts were activated with CCL21 (200 ng/mL) (F) or anti-CD3 for the indicated times and lysed. Lysates were then analyzed with the indicated antibodies to measure JNK phosphorylation. Numbers indicate the relative abundance of normalized pJNK to JNK, in comparison to WT cells. The bar graph shows the quantification of these measurements. Results from one of three independent experiments are shown. (G) WT and Pak1(T)^−/− CD4⁺ T blasts were activated with CCL21 (200 ng/mL) for the indicated times and lysed. Lysates were then analyzed with the indicated antibodies to measure AKT phosphorylation. AKT phosphorylation levels were normalized to AKT. Numbers indicate the relative abundance of pAKT T308 and S473 in comparison to WT cells. The bar graph shows the quantification of these measurements. Results from one of five independent experiments are shown. Statistical analysis: unpaired Student's t-test (B,D–G). ns, not significant; *P < 0.05; **P < 0.01.

Figure 4

Pak1 regulates L-selectin shedding in activated CD4⁺ T cells. (A) Analysis by ELISA of soluble L-selectin in the supernatants of WT and Pak1(T)^−/− T cell blasts. Data were pooled from six independent experiments. (B) Representative time-dependent calcium flux (indo-violet/indo-blue) of Indo-1AM loaded WT and Pak1(T)^−/− CD4⁺ T cell blasts, stimulated with CCL21 (200 ng/mL) for the indicated time. Results from one of three independent experiments are shown. (C) Calmodulin (CaM) was immunoprecipitated and eluted material was blotted for co-precipitation L-selectin and CaM in lysates prepared from WT and Pak1(T)^−/− T cell blasts. Whole-cell lysates were blotted for CaM. Results from two independent experiments are shown. (D) Analysis of soluble L-selectin in the supernatant of WT and Pak1(T)^−/− CD4⁺ T cell blasts pre-treated for 20 min with DMSO (1:1,000), BAPTA-AM 10 μM and EGTA 2 mM, or TMI1 (0.5 μM), and incubated for 30 min in the presence of 100 ng/mL CCL21. (E) Left, L-selectin surface marker expression as measured by flow cytometry from WT and Pak1(T)^−/− CD4⁺ T cell blasts cultured with or without TMI1(10 μM) for 6 days. The results are representative of four experiments. Right, bar chart shows MFI (%) of L-selectin in WT and Pak1(T)^−/− blast T cells. (F) Percentage of WT, Pak1(T)^−/− and Pak1(T)^−/− treated with TMI1 cells per field of view. Statistical analysis: unpaired Student's t-test (A); ANOVA between selected columns with Sidak's multiple comparison test (D); ANOVA with Tukey's multiple comparison test (E,F). ns, not significant; *P < 0.05; **P < 0.01.