Regulation of thymocyte positive selection and motility by GIT2

Journal name:
Nature Immunology
Volume:
11,
Pages:
503–511
Year published:
DOI:
doi:10.1038/ni.1868
Received
Accepted
Published online

Abstract

Thymocytes are highly motile cells that migrate under the influence of chemokines in distinct thymic compartments as they mature. The motility of thymocytes is tightly regulated; however, the molecular mechanisms that control thymocyte motility are not well understood. Here we report that G protein–coupled receptor kinase-interactor 2 (GIT2) was required for efficient positive selection. Notably, Git2−/− double-positive thymocytes showed greater activation of the small GTPase Rac, actin polymerization and migration toward the chemokines CXCL12 (SDF-1) and CCL25 in vitro. By two-photon laser-scanning microscopy, we found that the scanning activity of Git2−/− thymocytes was compromised in the thymic cortex, which suggests GIT2 has a key role in regulating the chemokine-mediated motility of double-positive thymocytes.

At a glance

Figures

  1. Impaired generation of CD4SP thymocytes in DO11.10+ Git2-/- mice.
    Figure 1: Impaired generation of CD4SP thymocytes in DO11.10+Git2−/− mice.

    (a) Flow cytometry analysis of the expression of CD4 and CD8 on thymocytes from DO11.10+ wild-type (Git2+/+) mice and DO11.10+ Git2−/− mice to assess stages of thymocyte development. Numbers in quadrants indicate percent cells in each. (b) Cell numbers of thymic subsets from DO11.10+ wild-type and DO11.10+ Git2−/− mice (n = 10–11 mice per genotype). (c) Expression of the TCR transgene in total thymocytes from DO11.10+ wild-type or DO11.10+ Git2−/− mice, assessed with the transgene-specific antibody KJ1-26. Numbers above bracketed lines indicate percent KJ1-26+ cells. (d) Frequency of KJ1-26hiCD69hi cells among DP thymocytes (left) and number of KJ1-26hiCD69hi DP cells (right) from DO11.10+ wild-type or DO11.10+ Git2−/− mice (n = 3 mice per genotype). (e) Expression of KJ1-26 in CD4+ T cells from DO11.10+ wild-type spleen (n = 3) or DO11.10+ Git2−/− spleen (n = 5). Numbers above bracketed lines indicate percent KJ1-26+ cells. (f) Frequency of CD4+KJ1-26+ cells in the periphery of DO11.10+ wild-type mice (n = 3) and DO11.10+ Git2−/− mice (n = 5). *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001 (unpaired two-tailed Student's t-test). Data are representative of nine (ac) or three (df) experiments (error bars (b,d,f), s.e.m.).

  2. Fewer CD4SP thymocytes in TCR-transgenic Git2-/- mice because of impaired positive selection, not more apoptosis or negative selection.
    Figure 2: Fewer CD4SP thymocytes in TCR-transgenic Git2−/− mice because of impaired positive selection, not more apoptosis or negative selection.

    (a) Intracellular staining of BrdU+ cells after 48 h of BrdU incorporation by DP or TCR-transgenic CD4SP thymocytes from TCR-transgenic wild-type or Git2−/− mice, presented as a percentage of total live thymocytes. Vα2+ indicates cells positive for the OT-II transgene; KJ1-26+ indicates cells positive for the DO11.10 transgene. *0.01 < P < 0.05; **P < 0.001 (unpaired two-tailed Student's t-test). Data are pooled from two independent experiments (OT-II+ mice: wild-type, n = 8; Git2−/−, n = 6) or are from a single experiment (DO11.10+ mice: n = 3 per genotype; error bars, s.e.m.). (b) Apoptosis of DO11.10+ wild-type or DO11.10+ Git2−/− thymocytes, analyzed by staining with 7-amino-actinomycin D and annexin V at 0, 4 and 24 h of incubation in medium containing 10% FBS. Data are representative of two independent experiments with two to three mice per genotype each (error bars, s.e.m.). (c) Intracellular staining of active caspase-3 in DO11.10+ wild-type and DO11.10+ Git2−/− thymocytes. Data are pooled from two independent experiments with five mice per genotype (error bars, s.e.m.). (d) Frequency of Vβ3+, Vβ5+ and Vβ11+ cells among CD4SP thymocytes or CD4+ T cells from lymph nodes from wild-type and Git2−/− mice on a BALB/c background (n = 3 per genotype). Data are representative of three experiments (error bars, s.e.m.).

  3. The defect in the positive selection of TCR-transgenic Git2-/- thymocytes is intrinsic to hematopoietic cells.
    Figure 3: The defect in the positive selection of TCR-transgenic Git2−/− thymocytes is intrinsic to hematopoietic cells.

    (a) Flow cytometry analysis of the generation of CD4SP thymocytes by wild-type BALB/c hosts reconstituted with DO11.10+ wild-type donor bone marrow (DO11.10+ Git2+/+Git2+/+) or DO11.10+ Git2−/− donor bone marrow (DO11.10+ Git2−/−Git2+/+). Data are representative of three independent experiments with five chimeras. (b) Flow cytometry analysis of the generation of CD4SP thymocytes in wild-type and Git2−/− hosts reconstituted with DO11.10+ wild-type bone marrow. Data are representative of a single experiment with five chimeras per condition. In a,b, numbers in outlined areas indicate percent cells in each; numbers above bracketed lines indicate percent KJ1-26+ cells. (c) Thymic architecture of chimeras reconstituted with DO11.10+ wild-type or DO11.10+ Git2−/− bone marrow cells. Original magnification, ×4. (d) DP-to-CD4SP transition of thymocytes generated from OT-II+ wild-type or OT-II+ Git2−/− bone marrow hematopoietic stem cells in a mixed–bone marrow chimera reconstituted with a 1:1 mixture of bone marrow from two donor groups with different congenic markers (OT-II+ wild type (CD45.1+CD45.2+) and OT-II+ Git2−/− CD45.2+)). NS, not significant; *0.01 < P < 0.05; **P < 0.001 (unpaired two-tailed Student's t-test); P values below graph compare wild-type with Git2−/−. Data are pooled from seven independent experiments (mean and s.e.m.).

  4. Greater migratory activity of Git2-/- DP thymocytes in response to SDF-1 or CCL25.
    Figure 4: Greater migratory activity of Git2−/− DP thymocytes in response to SDF-1 or CCL25.

    (a) Transwell migration of wild-type and Git2−/− thymocytes in the presence or absence (Media) of SDF-1 or CCL25, quantified by flow cytometry after staining with anti-CD4 and anti-CD8 and presented as the percentage of input cells that migrated to the lower chamber. Data are from five independent experiments (means and s.e.m. of duplicate assays). (b) Flow cytometry analysis of the expression of CD69 and TCRβ (top) or CD69 (bottom) in DP populations of input or migrated wild-type or Git2−/− thymocytes in the presence of CCL25 or SDF-1; thymocytes were stained with anti-CD4, anti-CD8, anti-CD69 and anti-TCRβ. Numbers in quadrants (top) indicate percent TCRβCD69 cells (left) or TCRβ+CD69+ cells (right); numbers above bracketed lines (bottom) indicate percent cells. Data are representative of five experiments. (c) Migration of preselection CD69loTCRβlo (top) and post-selection CD69hiTCRβhi DP thymocytes (bottom). Data are from three independent experiments (error bars, s.e.m. of duplicate assays). *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001 (unpaired two-tailed Student's t-test).

  5. Greater directional and random motility of Git2-/- thymocytes.
    Figure 5: Greater directional and random motility of Git2−/− thymocytes.

    (a) Transwell migration of wild-type and Git2−/− thymocytes from the upper well in the absence of SDF-1 (Media), or with SDF-1 in the top well (Top), the bottom well (Bottom) or both wells (Both). Data are representative of two independent experiments (error bars, s.e.m.). (b) Directional migration of wild-type and Git2−/− thymocytes on two-dimensional surfaces, monitored by time-lapse fluorescence microscopy with alginate beads; thymocytes were labeled with different fluorescent dyes, a 1:1 mixture of thymocytes in a solution of collagen (1.5 mg/ml) was placed on chambered coverglass coated with ICAM-1 (1 μg/ml) in the presence of unloaded control beads (left) or SDF-1-loaded beads (right) and cells were imaged for 40 min to 1 h at intervals of 30 s. Each symbol represents the track of one thymocyte; small horizontal lines indicate the mean. *P = 0.03 (unpaired two-tailed Student's t-test). Data are representative of two (control beads) or three (SDF-1-releasing beads) independent experiments (mean and s.e.m. in plots). (c) Average migration speed of wild-type and Git2−/− thymocytes in the presence of control beads or SDF-1-loaded beads as in b, binned into intervals of 1 μm/min; the frequency of total cells migrating in each range is normalized as a percentage of the maximum (% of max). Data are representative of two (control beads) or three (SDF-1-releasing beads) independent experiments. (d) Directionality index of wild-type and Git2−/− thymocytes that migrated more than 20 μm toward SDF-1-releasing beads (left) and average migration speeds of wild-type and Git2−/− thymocytes that moved toward SDF-1-releasing beads by more than 20 μm, binned into intervals of 1 μm/min and presented as in c (right; cells that did not move toward SDF-1-releasing beads by more than 20 μm are excluded). Each symbol (left) represents one thymocyte; small horizontal lines indicate the mean. NS, unpaired two-tailed Student's t-test. Data are representative of three independent experiments.

  6. More Rac1 activation and actin polymerization in Git2-/- thymocytes in response to SDF-1.
    Figure 6: More Rac1 activation and actin polymerization in Git2−/− thymocytes in response to SDF-1.

    (a) Immunoblot analysis (IB) of Rac1 activation in wild-type or Git2−/− thymocytes after 0, 0.5, 2, 5 and 15 min of SDF-1 stimulation (left), and increase in Rac1 activation normalized to GTP-Rac1 in the resting state (right). Precip, precipitation (with PAK1-PBD agarose); WCL, whole-cell lysates (control). (b) F-actin polymerization in Git2−/− DP thymocytes (left) and CD4SP thymocytes (right) in response to SDF-1, assessed by staining with phalloidin–Alexa Fluor 488, anti-CD4 and anti-CD8; the mean fluorescence intensity of phalloidin–Alexa Fluor 488 staining is normalized to that of wild-type cells at rest. Data are from three independent experiments per condition (error bars, s.e.m.).

  7. More Git2-/- DP thymocytes overcome TCR-mediated stop signals in the presence of SDF-1.
    Figure 7: More Git2−/− DP thymocytes overcome TCR-mediated stop signals in the presence of SDF-1.

    (a) Transwell migration of wild-type and Git2−/− DP thymocytes across filters coated with BSA or ICAM-1 (3 μg/ml), in response to media or SDF-1 in the bottom well; migration was quantified by flow cytometry after staining with anti-CD4 and anti-CD8 and is presented as migrating cell number/input cell number. (b) Transwell migration of wild-type and Git2−/− DP thymocytes across filters coated with ICAM-1 (3 μg/ml) plus either of two concentrations of anti-CD3 (5 μg/ml or 20 μg/ml), in response to media or SDF-1 in the bottom well. *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001 (unpaired two-tailed Student's t-test). Data are from two independent experiments (error bars, s.e.m. of duplicate assays).

  8. Altered motility of Git2-/- thymocytes in the cortex.
    Figure 8: Altered motility of Git2−/− thymocytes in the cortex.

    (a) Wild-type (blue; CFP) and Git2−/− (green; GFP) thymocytes from the thymus of a partial mixed–bone marrow chimera at a single time point, superimposed on cell tracks (top); red, blood vessels. Below, trajectories of individual cells, presented as tracks: yellow, CFP+ wild-type thymocytes; purple, GFP+ Git2−/− thymocytes. Scale bars, 20 μm. Data are representative of one experiment (wild type) or three experiments (Git2−/−). (b) Average migration speed of wild-type and Git2−/− thymocytes. Each symbol represents an individual tracked thymocyte; green horizontal lines indicate the mean (small black horizontal lines, s.e.m.). *P < 0.0001 (unpaired two-tailed Student's t-test). Data are compiled from one experiment (wild type) or three experiments on 2 different days (Git2−/−). (c) Directionality index of wild-type and Git2−/− thymocytes, plotted as a function of average speed (0–10 μm/min), binned as intervals of 2–3 μm/min, 3–4 μm/min or 4–5 μm/min (gray vertical lines); each symbol represents an individual tracked thymocyte; numbers above plots indicate average directionality index of each bin. *P = 0.0004 for 2–3 μm/min; **P < 0.0001 for 3–4 μm/min and for 4–5 μm/min (unpaired two-tailed Student's t-test). Data are compiled from one experiment (wild type) or three experiments on 2 different days (Git2−/−). (d) Fluorescence microcopy of the migration of Git2−/− thymocytes near blood vessels, presented as a projection of single time point with cell tracks superimposed. Scale bar, 10 μm. Data are representative of three experiments. (e) Immunofluorescence of thymic sections from wild-type mice injected with lectin–fluorescein isothiocyanate (to label blood vessels) and stained with anti-SDF-1, showing SDF-1+ blood vessels. Original magnification, ×20 (top) or ×40 (bottom). Data are representative of two experiments.

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Author information

Affiliations

  1. Department of Medicine, Division of Rheumatology, Rosalind Russell Medical Research Center for Arthritis, University of California San Francisco, San Francisco, California, USA.

    • Hyewon Phee,
    • Marianne Mollenauer &
    • Arthur Weiss
  2. Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California Berkeley, Berkeley, California, USA.

    • Ivan Dzhagalov &
    • Ellen Robey
  3. Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California, USA.

    • Marianne Mollenauer &
    • Arthur Weiss
  4. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Yana Wang
  5. Department of Biological Engineering and Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Darrell J Irvine
  6. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.

    • Darrell J Irvine

Contributions

H.P. designed the study, did experiments, analyzed data and wrote the manuscript; M.M. did experiments; I.D. did the two-photon experiments and analyzed data; Y.W. and D.J.I. supplied alginate beads; E.R. supervised the two-photon experiments and discussed data; and A.W. designed the study, supervised the research and revised the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (4M)

    Supplementary Figures 1–8, Table 1 and Methods

Movies

  1. Supplementary Video 1 (3M)

    Directional migration of wild-type and Git2−/− thymocytes to SDF-1.

  2. Supplementary Video 2 (948K)

    Migration of wild-type and Git2−/− thymocytes in intact thymic lobes using two-photon microscopy.

  3. Supplementary Video 3 (1M)

    Accumulation and movement of Git2−/− thymocyte on small blood vessels.

  4. Supplementary Video 4 (288K)

    Migratory behavior of wild-type thymocytes that move away from small blood vessels.

Additional data