A Requirement of Protein Geranylgeranylation for Chemokine Receptor Signaling and Th17 Cell Function in an Animal Model of Multiple Sclerosis

Abstract

Precisely controlled lymphocyte migration is critically required for immune surveillance and successful immune responses. Lymphocyte migration is strictly regulated by chemokines and chemokine receptors. Here we show that protein geranylgeranylation, a form of post-translational protein lipid modification, is required for chemokine receptor-proximal signaling. Mature thymocytes deficient for protein geranylgeranylation are impaired for thymus egress. Circulating mature T cells lacking protein geranylgeranylation fail to home to secondary lymphoid organs or to transmigrate in response to chemokines in vitro. Mechanistically, protein geranylgeranylation modifies the γ-subunits of the heterotrimeric small GTPases that are essential for chemokine receptor signaling. In addition, protein geranylgeranylation also promotes the differentiation of IL-17-producing T helper cells while inhibiting the differentiation of Foxp3⁺ regulatory T cells. Finally, mice with T cell lineage-specific deficiency of protein geranylgeranylation are resistant to experimental autoimmune encephalomyelitis induction. This study elucidated a critical role of protein geranylgeranylation in regulating T lymphocyte migration and function.

Keywords: T cells; adaptive immune response; autoimmunity; lymphocyte migration; protein geranylgeranylation.

Figure 1

Defective mature thymocytes egress in Pggt1b^fl/fl dLckCre mice (A) Flow cytometry analysis of thymic CD4 SP and CD8 SP cell expression of CD69 and CD62L and the expression of S1P1R based on sub-populations defined by CD62L^low CD69^high, CD62L, and CD69^intermediate or CD62L^high CD69^low; (B) Total number of thymocytes; (C) the percentage and number of CD4 SP and CD8 SP of total thymocytes; (D) percentage and the total number of CD4 SP and CD8 SP subpopulations described in (A). Each dot in the graphs (B–E) represents a single mouse (n.s. statistically not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t-test).

Figure 2

T-Lympopenia in secondary lymphoid organs of Pggt1b^fl/fl dLckCre mice (A) Flow cytometry analysis of CD19 and TCRβ positive cells in the blood, spleen, and lymph nodes; (B–G) Total cell number of lymphocytes (B); Percentage (C) and number (D) of TCRβ⁺ cells; Total number of CD4⁺ (E), CD8⁺ (F), and CD19⁺ (G) cells in blood, spleen, and lymph nodes. Each dot represents a single mouse iLN, abLN, mLN: inguinal, axillary, and brachial, mesenteric lymph nodes, respectively (n.s. statistically not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t-test).

Figure 3

Defective in vitro transmigration and in vivo homing of Pggt1b-deficient CD4⁺ T cells. (A) Flow cytometry analysis of CD4⁺, CD8⁺ T cells in transwell migration assay in response to CCL21; (B,C) percentage of input CD4⁺ (B) and CD8⁺ (C) cells migrated into the low chamber of transwell in response to CCL21; (D) Flow cytometry analysis of eFluor450-labelled CD4⁺ naive T cells from Pggt1b^fl/fl mice or Far Red-labelled naive CD4⁺ T cells from Pggt1b^fl/fl dLckCre mice that were mixed at a ratio of 1:1 before injected i.v. into recipient mice; (E,F) Percentage of CD4⁺ T cells out of the total cells injected in the blood, spleen (E) and various lymph nodes (F) of the recipient mice. Each dot represents a single mouse. a/b, axillary and brachial; c, cervical; i, inguinal; m, mesenteric lymph nodes (n.s. statistically not significant; *p < 0.05, **p < 0.01, ***p < 0. 001, unpaired t-test).

Figure 4

Protein geranylgeranylation is required for heterotrimeric small G-protein mediated GPCR-proximal signaling. (A) Western blot analysis of CCL20-induced phosphorylation of Akt and Erk in Th17 cells and density of phosphor-Akt calculated using ImageJ; (B) FTase I and GGTase I scores of the 12 γ-subunits of the small heterotrimeric GTPases calculated using an algorithm described in the text; (C) Fold change of the expression of the genes encoding the 12 γ-subunits of heterotrimeric small GTPase in naive and effector CD4⁺ T cells analyzed by qRT-PCR; (D) Flow cytometry analysis of EGFP expression in Th17 cells infected with retrovirus carrying cDNAs encoding mutant γ-subunits capable of being farnesylated; (E) Western blot analysis of phosphor-Akt and phosphor-Gsk3βS9 in Th17 cells described in (D); (F) Density of phosphor-Akt and phosphor-Gsk3β in (E) calculated using Image J (Results are representatives of three biologically independent experiments, n.s. statistically not significant; **p < 0.01, ***p < 0. 001, ****p < 0. 0001unpaired t-test).

Figure 5

Pggt1b^fl/fl dLckCre mice are resistant to EAE induction. (A) Clinical scores of mice immunized with MOG35-55 peptide emulsified in complete Freud's adjuvant; (B–D) Flow cytometry analysis of spinal cords leukocytes isolated from mice on day 19 after immunization and stained with antibodies against CD45, CD11b, CD4, Ly6C, Ly6G, CD44, CD64, and MHC II and gated according to a strategy described in the text to distinguish myeloid, lymphoid, microglia, CD4⁺ T cells, and monocyte-derived dendritic cells (MoDCs); (E) Percentage of lymphoid, myeloid, microglia, CD4⁺ T cells, and MoDCs in the spinal cord (Results are from two independent biological experiments with a total of 20 mice (10 male, 10 female)) [NS, not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001, way anova (A), unpaired t-test (E)].

Figure 6

Defective effector T cell egress from secondary lymphoid organs after primary immunization in Pggt1b^fl/fldLckCre mice. (A) Flow cytometry analysis of effector (CD44^high, CD62L ^low) CD4⁺ T cells in the spleen, draining lymph nodes and blood 7 days after immunization; (B) Flow cytometry analysis of MOG-specific CD4⁺ effector T cells in spleen, lymph nodes and blood 7 days after immunization; absolute number of CD4⁺ effector cells and MOG-specific CD4⁺ effector T cells in spleen (C), lymph nodes (D), and blood (E). Results are representative of two biologically independent experiments with a total of 16 (8 male, 8 female) mice (NS, not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001 unpaired t-test).

Figure 7

Pggt1b-deficient naive T cells are predisposed to differentiate into T regulatory cells in vitro. (A) Flow cytometry analysis of the expression of Foxp3 and IL-17A in cells cultured in the presence of plate-bound anti-CD3 and anti-CD28 antibodies and TGFβ1 and IL-6 for 72 h; (B) Percentage of IL-17A+ or Foxp3+ CD4+ cells in the culture described in (A); (C) Fold change of Th17 signature cytokines in cells described in (A) analyzed by qRT-PCR; (D) Flow cytometry analysis of GM-CSF⁺ and IL-17⁺ CD4⁺ T cells in cells cultured in the presence of TGFβ1 and IL-6, IL-1, IL-6, and IL-23 or IL-1 IL-23; (E) Percentage of GM-CSF⁺ and IL-17A⁺ CD4⁺ T cells in cultures described in (D); (F) Flow cytometry analysis of naive T cells 72 h after cultured in the presence of plate-bound anti-CD3 and anti-CD28 antibody and different concentrations of TGFβ1; (G) percentage of Foxp3+ CD4+ T cells in the culture described in (F) and qRT-PCR analysis of the expression T regulatory cell genes in cell cultures described in (F). Results are representative of three independent biological experiments [n.s. statistically not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way anova (C), unpaired t-test (G)].