Formin-like 1 mediates effector T cell trafficking to inflammatory sites to enable T cell-mediated autoimmunity
- Department of Biomedical Research, National Jewish Health, United States
- Department of Immunology and Microbiology, University of Colorado School of Medicine, United States
- Barbara Davis Center, University of Colorado School of Medicine, United States
Figures
Figure 1 with 3 supplements
Fmnl1 targeting strategy and knock-out confirmation.
(A) Schematic of the targeted Fmnl1 allele after homologous recombination with the targeting construct. (B) Representative western blot showing complete loss of FMNL1 protein expression in T cells …
Figure 1—figure supplement 1
PCR confirmation of targeting vector insertion.
(A) PCR reaction (with GF4 and LAR3 primers, see Figure 1A) showing ES cell clones positive for the insertion at the 5’ end of the targeting construct. (B) PCR reaction (with RCF and GR4 primers, …
Figure 1—figure supplement 2
T cell development and lymphocyte populations in peripheral lymphoid organs are not altered in FMNL1 KO Mice.
(A) Thymic populations and T cell development are not altered in FMNL1 KO mice. Representative CD4 by CD8 flow cytometry plots of single cell gated populations from the thymus of WT or FMNL1 KO …
Figure 1—figure supplement 3
Myeloid populations in peripheral lymphoid organs are not altered in FMNL1 KO mice.
(A) Blood myeloid populations are not altered in FMNL1 KO mice. Frequencies of singlet gated cells (left) and total numbers of the indicated populations per ml of blood (right). (B) Splenic myeloid …
Figure 2 with 1 supplement
FMNL1-deficient T cells are impaired in trafficking to the pancreatic islets and inducing type 1 diabetes.
(A) Experimental set-up to quantify activated T cell trafficking to the inflamed pancreatic islets. To induce inflammation of the pancreatic islets, 7 days prior to harvest, WT OT-I transgenic T …
Figure 2—figure supplement 1
FMNL1 deficiency does not impair ex vivo T cell activation.
T cells from WT or FMNL1 KO mice were isolated and then activated ex-vivo with anti-CD3 and CD28 antibodies in the presence of WT irradiated, congenically marked, CD45.1+ splenocytes. After 2 days, …
Figure 3 with 2 supplements
FMNL1-deficient T cells are impaired in trafficking to the CNS and inducing EAE.
(A) Experimental set-up to quantify activated T cell trafficking to the inflamed CNS. Activated, differentially dye-labeled, polyclonal CD45.2/.2 WT and FMNL1 KO T cells were co-transferred …
Figure 3—figure supplement 1
FMNL1 deficiency does not impair ex vivo T cell cognate peptide stimulation.
CD4+ T cells from WT or FMNL1 KO 2D2 TCR transgenic mice were isolated and then activated ex-vivo with MOG peptide in the presence of WT irradiated, congenically marked, CD45.1+ splenocytes. After 2 …
Figure 3—figure supplement 2
FMNL1 deficiency does not impair T cell trafficking to lymphoid tissues.
(A) Experimental set-up to quantify naive T cell trafficking to lymphoid organs. Differentially fluorescent dye-labeled naive WT and FMNL1 KO T cells were co-transferred intravenously at a 1:1 ratio …
Figure 4
FMNL1 deficiency impairs T cell TEM at the diapedesis step.
Ex vivo activated CFSE or CellTrace Yellow (CTY) dye-labeled T cells were perfused into flow chambers containing bEnd.3 brain endothelial cell monolayers and kept under shear flow (2 dyne/cm2) for …
Figure 5 with 1 supplement
FMNL1 promotes nucleus transmigration in T cells undergoing TEM.
TEM of dye-labeled WT T cells was set up as in Figure 4. After 5 min of TEM under flow cells were fixed, permeabilized, and stained with DAPI and an anti-FMNL1 antibody (A) Representative maximum Z …
Figure 5—figure supplement 1
Immunofluorescent FMNL1 antibody staining is specific for FMNL1.
TEM under flow of differentially dye-labeled WT and FMNL1 KO T cells was set up as in Figure 4. After 5 min of TEM under flow cells were fixed, permeabilized, and stained with a mouse monoclonal …
Figure 6 with 2 supplements
T cell migration through narrow pores is FMNL1 dependent.
(A) FMNL1-deficient T cells are impaired in chemotaxis through narrow pores. Percentage of activated cells migrating through transwell membranes of the indicated pore size in response to CXCL12. (B) …
Figure 6—figure supplement 1
Transwell migration and actin polymerization responses to CXCL10.
(A) FMNL1-deficient T cells are impaired in chemotaxis through narrow pores. Percentage of activated cells migrating through transwell membranes of the indicated pore size in response to CXCL10. (B) …
Figure 6—figure supplement 2
Re-expression of FMNL1 in KO cells.
Representative western blot for Figure 6F showing expression levels of FMNL1 in FMNL1 KO or WT T cells transduced with FMNL1-expressing or control retroviral constructs. Tubulin staining is shown as …
Figure 7
FMNL1 mediates posterior perinuclear actin polymerization to promote T cell migration through environmental constrictions.
Ex vivo activated control LifeAct-GFP and FMNL1 KO LifeAct-GFP T cells were stained with Hoechst, added to PDMS microchannels and imaged by spinning-disk confocal time-lapse microscopy. (A) …
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Figure 7—source data 1
Data points for the graphs in Figure 7D are provided as an Excel spreadsheet.
- https://cdn.elifesciences.org/articles/58046/elife-58046-fig7-data1-v2.xlsx
Videos
Video 1
Example of a WT control T cell undergoing transendothelial migration imaged by time-lapse confocal microscopy.
Control and FMNL1 KO T cells were activated ex-vivo and differentially dye-labeled with CFSE or CellTrace Yellow (CTY). These T cells were mixed at a 1:1 ratio and perfused into flow chambers …
Video 2
Example of a FMNL1 KO T cell unable to complete transendothelial migration.
Control and FMNL1 KO T cells were activated ex-vivo and differentially dye-labeled with CFSE or CellTrace Yellow (CTY). These T cells were mixed at a 1:1 ratio and perfused into flow chambers …
Video 3
Example of a control LifeAct-GFP T cell displaying posterior perinuclear actin polymerization while migrating through a microchannel constriction.
Control LifeAct-GFP T cells were activated ex-vivo, labeled with Hoechst to visualize their nuclei, and added to microchannel devices. T cells crawling in the microchannels were imaged at 37°C for …
Video 4
Example of a FMNL1 KO/LifeAct-GFP T cell failing polymerize actin at the back of the cell and unable to migrate through a microchannel constriction.
FMNL1 KO/LifeAct-GFP T cells were activated ex-vivo, labeled with Hoechst to visualize their nuclei, and added to microchannel devices. T cells crawling in the microchannels were imaged at 37°C for …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic Reagent (M. musculus) | FMNL1 KO | This Paper | FMNL1 deficient mouse on C57BL/6 genetic background | |
| Genetic Reagent (M. musculus) | LifeAct-GFP | Riedl et al., 2008 | RRID:IMSR_EM:12427 | Mice expressing LifeAct-GFP construct on C57BL/6 genetic background |
| Antibody | anti-FMNL1 (mouse monoclonal) | Santa Cruz Biotechnology | Cat#: sc-390466 RRID:AB_2847962 Clone A-4 | IF (1:100) |
| Recombinant DNA Reagent | FMNL1-IRES-GFP MMLV (plasmid) | VectorBuilder | pMMLV vector that expresses FMNL1 and GFP (under an IRES) | |
| Recombinant DNA Reagent | Control GFP MMLV and Control RFP MMLV (plasmids) | VectorBuilder | Control pMMLV vectors that express GFP or RFP | |
| Other | Microchannel fluidic devices | 4DCell | Cat#s: MC011 and MC019 | Microchannel devices with variable channel widths and constrictions |
| Cell line (M. musculus) | bEnd.3 | ATCC (Montesano et al., 1990) | Cat#: CRL-2299 RRID:CVCL_0170 | Mouse endothelial cell line used in the TEM assays |
