T cell–specific dynamin 2 deficiency leads to lymphopenia and accumulation of mature thymocytes. (A and B) Flow cytometric analysis of blood CD4 and CD8 T cells (A) and numbers of CD4 T cells (B) in blood, spleen, and lymph node from Dnm2 HET and KO mice (n = 10). (C and D) Flow cytometric analysis (C) and numbers (D) of double-negative (DN), DP, and CD4 and CD8 SP thymocytes from Dnm2 HET and KO mice (n = 13). (E and F) Flow cytometric analysis (E) and numbers (F) of immature (TCRβ^hiCD24^hi) and mature (TCRβ^hiCD24^lo) CD4 SP thymocytes from Dnm2 HET and KO mice (n = 13). Horizontal bars indicate the mean. (G) Bone marrow cells from Dnm2 HET or KO mice (CD45.2⁺) were mixed 1:1 with WT B6 cells (CD45.1⁺) and injected into irradiated Rag1 KO recipients (CD45.1⁺) to generate bone marrow chimeras. Ratio of CD4 and CD8 T cells in thymus (THY), lymph nodes (LN), spleen (SPL), and blood (BL) from Dnm2 HET/B6 and Dnm2 KO/B6 mixed chimeras (n = 16–17) is shown. (H) Mixed bone marrow chimeras were generated as in G. Ratio of immature and mature CD4 and CD8 SP thymocytes from Dnm2 HET/B6 and Dnm2 KO/B6 mixed chimeras (n = 11–16) is shown. Ratios were normalized to the ratio of DP thymocytes to account for variable chimerism among mice. The dashed line indicates a control cell/KO cell ratio of 1. All error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by unpaired Student’s t test (B, D, F, and G) or one-way ANOVA, Tukey’s post-test (H). Results are representative of or combined from three (D and F), four (G and H), or more than five (A–C and E) experiments.

Dynamin 2 is required for T cell egress from thymus. (A) Mixed Dnm2 HET/B6 and Dnm2 KO/B6 bone marrow chimeras generated as in were injected intrathymically with 20 µg FITC to label CD4⁺ and CD8⁺ RTEs. RTEs were gated as CD4⁺ or CD8⁺FITC⁺ cells in blood, spleen, and lymph node 24 h after injection. Ratio of HET/B6 and KO/B6 FITC⁺ T cells in various tissues is shown (n = 11–12). Ratios in blood, spleen, and lymph nodes were normalized to the ratio in thymus (ratio = 1) to account for differences in labeling. (B and C) Mixed bone marrow chimeras generated as in were injected i.v. with 1 µg α–CD4-PE Ab. (B) Egressing WT CD4 SP thymocytes in circulation were gated as CD4-PE⁺ cells in untreated chimeras or chimeras injected with FTY720 (to inhibit egress) 16 h before analysis. (C) Ratio of egressing CD4 SP thymocytes (CD4-PE⁺) from Dnm2 HET/B6, Dnm2 KO/B6, and Cd4-creRosa26^PTX/+ (Rosa26-PTX)/B6 mixed bone marrow chimeras (n = 9–16). (A and C) The dashed line indicates a control cell/KO cell ratio of 1. All error bars represent SEM. **, P < 0.01; ***, P < 0.001 by one-way ANOVA, Tukey’s post-test (C) or two-way ANOVA, Bonferroni post-test (A). Results are combined from two (A) or six (B and C) experiments.

Dynamin 2 regulates S1PR1 signaling in vivo. (A) CD69 surface expression on thymocytes from Dnm2 HET and KO mice (n > 10) was measured by flow cytometry. (B) Quantitative RT-PCR analysis of CD69 mRNA expression in thymocyte populations from Dnm2 HET and KO mice (n = 4). mRNA expression was normalized to Hprt. (C) Flow cytometric analysis of CD69 surface expression on immature CD4 SP thymocytes treated with α-CD69 Ab in vitro (n = 6). MFI, mean fluorescence intensity. (D and E) Flow cytometric analysis (D) and frequencies (E) of S1PR1-CD69 coexpressing CD4 SP thymocytes from Dnm2 HET and KO mice (n = 24). Horizontal bars indicate the mean. (F) Flow cytometric analysis of S1PR1 and CD69 surface expression on naive (CD44^lo) CD4 T cells from Dnm2 HET and KO mice (n = 9). (G and H) Dnm2 HET and KO mice received 30 mg/liter DOP in the drinking water or water alone. 3 d later, CD69 surface expression on mature CD4 SP thymocytes (n = 7) was measured by flow cytometry. All error bars represent SEM. ***, P < 0.001 by unpaired Student’s t test. Results are representative of or combined from two (C, G, and H), three (F), four (B), or more than five (A, D, and E) experiments.

Dynamin 2 is necessary for ligand-induced S1PR1 internalization at low S1P concentrations. (A) S1PR1 surface expression on mature CD4 SP thymocytes and naive (CD44^lo) CD4 T cells from Dnm2 HET and KO cells (n > 10) was measured by flow cytometry. To define background S1PR1 staining, control mice were injected i.p. with FTY720 16 h before flow cytometry. (B) S1PR1 surface expression on splenic naive CD4 T cells from Dnm2 HET/B6 and Dnm2 KO/B6 mixed bone marrow chimeras (n = 6) generated as in . Green histograms represent background S1PR1 staining (thymocytes from control chimeras injected with FTY720). (C) Dnm2 HET and KO mice were injected i.p. with PBS or FTY720 (2 mg/kg body weight). S1PR1 surface expression on mature CD4 SP thymocytes (n = 6) was measured by flow cytometry 16 h later. Green histograms represent S1PR1 staining on DP thymocytes as negative control. (D and E) S1PR1 surface expression on mature CD4 SP thymocytes from Dnm2 HET and KO mice treated with PBS or the indicated concentrations (per body weight) of FTY720 16 h before analysis (n = 3). (F and G) Dnm2 HET and KO mice received 30 mg/liter DOP in the drinking water or water alone. 3 d later, S1PR1 surface expression on mature CD4 SP thymocytes (n = 7) was measured by flow cytometry. Green histograms represent background S1PR1 staining (thymocytes from mice injected with FTY720). (H) S1PR1 surface expression on mature CD4 SP thymocytes from Dnm2 HET and KO mice treated in vitro with the indicated concentrations of S1P (n = 3–7). All error bars represent SEM. *, P < 0.05; **, P < 0.01 by unpaired Student’s t test. Results are representative of or combined from two (B, F, and G), three (C–E and H), or four (A) experiments.

Effect of dynamin 2 deficiency on thymocyte chemokine receptors. (A) CCR7 and CXCR4 surface expression on mature CD4 SP thymocytes from Dnm2 HET and KO mice (n = 6). (B) Chemotaxis of mature CD4 SP thymocytes from Dnm2 HET and KO mice. Migration through 5-µM Transwells in response to 0.3 µg/ml CXCL12 or 1 µg/ml CCL21 was assessed by flow cytometry after 3 h (n = 5–9). (C) Mixed Dnm2 HET/B6 and Dnm2 KO/B6 bone marrow chimeras generated as in were treated with saline or 1 mg AMD3100. After 24 h of continuous treatment, CXCR4 surface expression on mature CD4 SP thymocytes was measured by flow cytometry. (A and C) Green histograms show isotype staining (backgrd). (D) Number of egressing WT (B6) CD4 SP thymocytes from mixed bone marrow chimeras treated with saline (n = 9) or AMD3100 (n = 10) as in C. (E) Ratio of egressing CD4 SP thymocytes from Dnm2 HET/B6 and Dnm2 KO/B6 mixed bone marrow chimeras treated with saline or AMD3100 (n = 5) as in C. The dashed line indicates a control cell/KO cell ratio of 1. All error bars represent SEM. *, P < 0.05; **, P < 0.01 by unpaired Student’s t test (B) or one-way ANOVA, Tukey’s post-test (E). Results are representative of or combined from two (A and C–E) or five (B) experiments.

Differential sensitivity of thymocyte-expressed GPCRs to ligand-induced internalization. (A and B) Thymocytes from WT mice were incubated with the indicated concentrations of S1P, CXCL12, or CCL21 in vitro. Flow cytometry was used to measure S1PR1, CXCR4, and CCR7 surface expression in response to their ligands S1P, CXCL12, and CCL21, respectively. (A) Representative histograms showing S1PR1 and CCR7 surface expression on mature CD4 SP thymocytes and CXCR4 expression on DP thymocytes. (B) Ligand-induced down-modulation of S1PR1, CXCR4, and CCR7 in response to their respective ligands (n = 6). Receptor down-modulation is shown as percent surface down-modulation (calculated as described in Materials and methods) to account for differences in staining intensity between receptors and to compare them in the same graph. Error bars represent SEM. Results are representative of or combined from two experiments.

S1PR1 internalization and CD69 down-modulation are not dependent on Gαi signaling. (A) S1PR1 surface expression on mature CD4 SP thymocytes and naive CD4 T cells (n = 5). Green histograms represent background S1PR1 staining (FTY720-treated mice). (B) WT and Cd4-creRosa26^PTX/+ mice were injected i.p. with PBS or FTY720 (2 mg/kg body weight). S1PR1 surface expression on mature CD4 SP thymocytes (n = 3) was measured by flow cytometry 16 h later. Green histograms represent S1PR1 staining on DP thymocytes as negative control. (C) S1PR1 and CD69 surface expression on CD4 SP thymocytes from WT and Cd4-creRosa26^PTX/+ mice (n = 4). Results are representative of two (B and C) or three (A) experiments.

Dynamin 2 is essential for sustained S1PR1 signaling and lymph node egress. (A and B) Thymocytes from Dnm2 HET and KO mice were treated with solvent, 100 nM S1P, or 1 µM SEW2871 (S1PR1 agonist) in vitro. After 1 and 5 min, intracellular pERM expression was measured in mature CD4 SP thymocytes by flow cytometry. (A) Representative histograms showing intracellular pERM expression in mature CD4 SP thymocytes. (B) pERM expression after stimulation with S1P or SEW2871 was normalized to solvent control. Symbols represent measurements from individual mice (n = 4–6). Horizontal bars indicate the mean. (C and D, top) Thymocytes from Dnm2 HET and KO mice were not exposed to S1P (no desensitization), exposed to low (10 nM; C) or high (1,000 nM; D) S1P concentration (desensitization), or exposed to S1P and washed/rested for 30 min after exposure to S1P (resensitization). S1PR1 surface expression on mature CD4 SP thymocytes was measured by flow cytometry. (bottom) Transwell migration of mature CD4 SP thymocytes from Dnm2 HET and KO mice to 50 nM S1P (n = 5). No desensitization, cells not preexposed to S1P; desensitization, cells preexposed to low (1–10 nM; C) or high (1,000 nM; D) S1P concentration; resensitization, cells preexposed to S1P, washed, and incubated in medium without S1P for 30 min before the Transwell assay. Migration was normalized to that of non-desensitized cells to allow comparison between different experiments. (E) Fluorescently labeled Dnm2 HET and KO splenocytes (CD45.2⁺) were cotransferred into B6 recipient mice (CD45.1⁺) to measure T cell egress from lymph nodes as described in Materials and methods. The fraction of transferred CD4 T cells remaining in lymph nodes 15 h after injection of Abs against integrin αL and α4 was measured by flow cytometry (n = 6). All error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by unpaired Student’s t test (E) or one-way ANOVA, Tukey’s post-test (B–D). Results are representative of or combined from two (A, B, and E) or three (C and D) experiments.

Normal primary chemotaxis of dynamin 2–deficient thymocytes to S1P in vitro. (A and B) Transwell migration of mature CD4 SP thymocytes from Dnm2 HET and KO mice (A) or from Dnm2 HET/B6 and Dnm2 KO/B6 mixed bone marrow chimeras (B) to S1P (n = 5). (C and D) cAMP amounts in thymocytes from Dnm2 WT and KO mice after incubation for 1 (C) and 5 min (D) in medium containing IBMX (phosphodiesterase inhibitor) and FSK (forskolin; adenylate cyclase activator) in the absence or presence of 10 nM S1P. All error bars represent SEM. Results are representative of or combined from two (B–D) or five (A) experiments.

S1PR1 overexpression rescues T cell egress in dynamin 2–deficient mice. (A and B) CD69 surface expression on mature CD4 SP thymocytes from Dnm2 HET, S1PR1 TG Dnm2 HET, Dnm2 KO, and S1PR1 TG Dnm2 KO mice (n = 6–9). (C and D) Flow cytometric analysis of CD4 SP thymocytes (C) and numbers of mature CD4 SP thymocytes (D) from Dnm2 HET, S1PR1 TG Dnm2 HET, Dnm2 KO, and S1PR1 TG Dnm2 KO mice (n = 9–12). All error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA, Tukey’s post-test. Results are representative of or combined from two (A and B) or three (C and D) experiments.