Layilin Regulates Treg Motility and Suppressive Capacity in Skin

Victoire Gouirand
Sean Clancy
Courtney Macon
Jose Valle
Mariela Pauli
Hong-An Troung
Jarish Cohen
Maxime Kinet
Margaret M Lowe
Samuel J Lord
Kristen Skruber
Hobart Harris
Esther Kim
Isaac Neuhaus
Karin Reif
Ali A Zarrin
Dyche R Mullins
Michael D Rosenblum author has email address

Department of Dermatology, UCSF, San Francisco, United States
TRex Bio, South San Francisco, United States
Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, United States
Department of Surgery, UCSF, San Francisco, United States

https://doi.org/10.7554/eLife.105277.1

Open access
Copyright information

Figures and data

Layilin is preferentially expressed on Tregs in skin and correlates with activation and motility signatures.
A-C. Single cells RNA-seq of FACS-purified CD45⁺ cells from healthy and psoriatic skin. n= 5 healthy skins and five psoriatic skins. A. Representative UMAP showing all clusters found. B. Dot plot of LAYN, FOXP3, and CD8A expression by clusters. C. UMAP of FOXP3 and LAYN expression in skin. D. UMAP of Tregs subset showing in red layilin non-expressing Tregs (LAYN<0) and blue layilin expressing Tregs (LAYN>0) based on gene count > 0. E-F. Violin plot of LAYN (E), FOXP3 and CTLA4 (F) in subset Tregs from skin followed by resclustering based on LAYN gene count > 0. G. Volcano plot of layilin expressing Tregs (right) compared to layilin non-expressing Tregs (left) obtained from pseudo bulk analysis performed on Tregs subset. H. Gene set enrichment analysis(GSEA) of top enriched pathways in layilin expressing Tregs compared to layilin non-expressing Tregs associated with panel F. I-J. TCR analysis run on Treg cluster subset by LAYN expression. In I, we show the relative abundance of TCR clonotype frequencies separated into five groups: rare being expressed between 0 and 10⁴, small between 10⁴ and 0.001, a medium between 0.001 and 0.01, large between 0.01 and 0.1, and hyperexpanded between 0.1 and 1. J. Representation of the top expressed clonotypes in subset of Tregs from skin resclustered according to LAYN gene count > 0.

Layilin does not affect Treg activation or suppression in vitro.
A. Schematic experimental design strategy starting with the isolation of human Tregs from peripheral blood, activated and expanded using anti-CD3/anti-CD28 stimulation and IL-2 for 12 days. We deleted LAYN using CRISPR/Cas9, and after four days, cells were used for the experiment. B. Frequency of LAYN⁺ Tregs gated on Live CD8^-CD4⁺CD25⁺FOXP3⁺ cells, and representative expression of layilin. n = 6 donors from two independent experiments. Significance was defined by a two-tailed Student’s t-test. *p < 0.05. C. Frequencies (on the left) and Mean Fluorescence Intensity (MFI, on the right) of sg-LAYN and sg-CTRL Tregs gated on Live CD8^-CD4⁺CD25⁺FOXP3⁺ cells expressing the different activation Treg markers listed on the x-axis. The top row graphs show results from cells stimulated with anti-CD3/anti-CD28; the bottom graphs show results obtained from cells without anti-CD3/anti-CD28 stimulation. n = 6 donors from two independent experiments. Significance was defined by using a multiple-paired t-test. D. Proliferation percentage measured using cell trace violet (CTV) for CD4⁺ T responder (left panel) or CD8⁺ T responder (right panel) after three days of suppression assay. Results are presented in the x-axis, and the different ratios of sg-LAYN and sg-CTRL Tregs and T responders used for the assay represent two independent experiments. Significance was defined by two-way ANOVA followed by Tukey’s multiple comparisons test.

Layilin limits Treg-mediated suppression of inflammation in vivo.
A. Experimental design strategy of the imiquimod (IMQ) model. After four days of tamoxifen injection, mice were rested for two days before the start of the IMQ treatment. They received a daily dose of IMQ on shaved back skin and were scored for erythema, scaling, skin lesions, and weight loss. After six days of treatment, the mice were euthanized. B. Representative pictures of mouse back skin after five days of treatment from each experimental group. C. Overall clinical score over time. n =7 mice from three independent experiments. Significance was defined by two-way ANOVA. ****p<0.0001. D. Absolute count of Teff, CD8, and Tregs obtained from each experimental group, respectively gated on Live CD3⁺TCRβ⁺ and CD4⁺FOXP3^- or CD8⁺, CD4⁺FOXP3⁺. n =7 and 6 mice per group from three independent experiments. Significance was defined by a two-tailed Student’s t-test. *p < 0.05. E. Absolute count of cells Teff, CD8, and Treg either IFNɣ+, IL17+, or TNFα⁺. n =7 and 6 mice per group from three independent experiments. Significance was defined by a multiple-paired t-test. *p < 0.05. F. Frequencies of Tregs activation markers gated on Live CD3⁺TCRβ⁺ CD4⁺FOXP3⁺ Tregs. n =7 and 6 mice per group from three independent experiments, except ICOS and CTLA from 2 mice and one experiment. Significance was defined by a multiple-paired t-test. G. Volcano plot of differential expression of genes from sorted Tregs gated on GFP⁺ from FOXP3-ERT2-Cre; Layn^fl/fl (on the right) or FOXP3-ERT2-Cre (on the left) after four days of treatment. Red dots represent differentially expressed genes; black dots are not differentially expressed ones. n = 5 mice per group from two independent experiments. H. Top deregulated pathways obtained from a GSEA comparing FOXP3-ERT2-Cre; Layn^fl/fl to FOXP3-ERT2-Cre Tregs. The top axis and bar plot represent the Normalized Enrichment Score (NES). The bottom axis and dot represent the adjusted p-value associated with pathways enrichment. n = 5 mice per group from two independent experiments.

Layilin enhances Treg adhesion and LFA-1 activation
A. The experimental design strategy starts with the isolation of human Tregs from peripheral blood, which were activated and expanded using anti-CD3/anti-CD28 stimulation and IL-2 for 12 days. We deleted LAYN using a CRISPR/Cas9; after four days, cells were sorted by flow cytometry based on LAYN expression, as represented by the representative histogram of LAYN expression on sgCtrl (stained with or without primary antibody detecting layilin followed by streptavidin-PE staining) or sgLayn Tregs, to obtain a pure population of LAYN⁺ or LAYN^- Tregs. After sort, cells were placed in culture for follow-up experiments presented in panels B, C and F. B. Volcano plot of differential genes expressed in LAYN⁺ (on the left) or LAYN^- (on the right) Tregs put in culture for 24 hours in the presence of anti-CD3/anti-CD28. Red dots represent differentially expressed genes; black dots are not differentially expressed ones. n = 4 paired donors for each group. C. Top deregulated pathways obtained from a GSEA analysis comparing LAYN⁺ to LAYN^- Tregs. The top axis and bar plot represent the Normalized Enrichment Score (NES). The bottom axis and dot represent the adjusted p-value associated with pathways enrichment. n = 4 paired donors for each group. D. Ratio of m24⁺ to total CD18⁺ Tregs gated on Live CD8^-CD4⁺CD25⁺FOXP3⁺ cells after three days culture with or without anti-CD3/anti-CD28 stimulation and in the presence of a layilin crosslinking antibody as described on the x-axis for 20 minutes during LFA-1 activation staining. n = 3 donors. Significance was defined by one-way ANOVA followed by Dunnet’s multiple comparisons test. *p < 0.05, ***p<0.001. E. Frequency of m24⁺ sg-LAYN and sg-CTRL Tregs gated on Live CD8^-CD4⁺CD25⁺FOXP3⁺ cells after 24 hours of culture in a non-coated plate or collagen IV coated plate. n = 6 donors from two independent experiments. Significance was defined by two-way ANOVA followed by Sidak’s multiple comparisons test. *p < 0.05. F. Numbers of cells attached to the bottom of the well are measured by the cell titer blue (expressed by relative fluorescence unit). n = 7 donors from two independent experiments. Significance was defined by two-way ANOVA. ns is non-significant, , ***p<0.001.

Layilin induces cytoskeleton changes in Tregs indicative of reduced motility.
A. Representative pictures of LAYN⁺ (on the top) or LAYN^- (on the bottom), Tregs phalloidin staining (in pink) and nuclei (in blue) after a 48-hour culture on collagen IV (left) or fibronectin (right) coated plates. White boxes highlight protuberances at the membranes. B. Quantification of phalloidin staining associated with pictures in A. n = 3 donors; ten pictures were quantified for each donor, and experiments were repeated independently twice. Significance was defined by two-way ANOVA followed by Sidak’s multiple comparisons test. *p < 0.05. C. Graphical hypotheses of layilin pathway mechanism of action. In the presence of layilin, it interacts with LFA-1 and maybe other integrins and stabilizes the formation of a focal adhesion complex, leading to the anchoring mechanism of the cells. Without layilin, integrins are less prone to adhesion mechanisms, and the ARP3 complex may act in lamellipodia formation, leading to more migratory Tregs.

A. Volcano plot comparing Treg cluster from psoriasis to Treg cluster from healthy skin. B. Quadrant representation of results from pseudo bulk RNAseq of Treg cluster subclustered in layilin expressing and non-expressing Tregs. Red dots represent differentially expressed genes; black dots are not differentially expressed ones. The left upper quadrant corresponds to genes enriched only in psoriatic layilin expressing Tregs, the right bottom quadrant are the ones only enriched in layilin expressing Tregs from healthy skin, and upper right quadrant corresponds to genes enriched in layilin expressing Tregs and common to both conditions.

A. qRT-PCR of layilin from sorted Tregs and Teff cells isolated respectively by gating on live CD45⁺CD3⁺CD4⁺CD8^-CD25^hiCD62L^hiGFP⁺ and CD45⁺CD3⁺CD4⁺CD8^-CD25^loCD62L^hiGFP^-. Tregs and Teff have been isolated from back skin of FOXP3-ERT2-Cre; Layn^fl/fl or FOXP3-ERT2Cre mice after four days of treatment. Each bar corresponds to 5 mice mixed to create a representative sample for each group. B. MFI of cells Teff, CD8, and Treg either IFNɣ+, IL17+, or TNFα⁺. n =7 and 6 mice per group from three independent experiments. Significance was defined by a multiple-paired t-test. C. MFI of Tregs activation markers gated on Live CD3⁺TCRβ⁺ CD4⁺FOXP3⁺ Tregs. n =7 and 6 mice per group from three independent experiments, except ICOS and CTLA from 2 mice and one experiment. Significance was defined by a multiple-paired t-test. D. Quality control of samples repartition used for bulk RNAseq in figure 5. E. Quality check of bulk RNAseq representing total of counts for each sample. In F and G, n = 5 mice per group from two independent experiments.

A. Quality check of bulk RNAseq representing total of counts for each sample (left panel) and layn count for each sample demonstrating CRISPR efficiency (right panel). n = 4 paired donors for each group. B. On the left panel, we represent the frequency of total m24+ Tregs gated on Live CD8-CD4+CD25+FOXP3+ cells after three days of culture with or without anti-CD3/anti-CD28 stimulation and in the presence of a layilin crosslinking antibody as described on the x-axis for 20 minutes during LFA-1 activation staining. The right panel represents the frequency of CD18⁺ Tregs. n = 3 donors. Significance was defined by one-way ANOVA followed by Dunnet’s multiple comparisons test. *p < 0.05, ***p<0.001. C. Frequency of LAYN⁺ Tregs gated on Live CD8^- CD4⁺CD25⁺FOXP3⁺ cells to measure CRISPR efficiency after 24 hours of culture in a non-coated plate or collagen IV-coated plate. n = 6 donors from two independent experiments.

Sign up for email alerts