Research ArticleINNATE IMMUNITY

PD-L1 expression on nonclassical monocytes reveals their origin and immunoregulatory function

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Science Immunology  21 Jun 2019:
Vol. 4, Issue 36, eaar3054
DOI: 10.1126/sciimmunol.aar3054
  • Fig. 1 PD-L1 selectively marks circulating NCMs.

    (A) Volcano plot showing gene expression profile in monocyte subsets. Difference of gene expression activity was calculated for each gene. A difference greater than +100% meant that a single gene was highly and only expressed in NCMs. Green box indicates genes that were significantly up-regulated in NCMs (P < 0.01; difference > +100%). (B) PD-L1 expression on blood leukocytes. Representative flow cytometry analysis (left) and quantification (right) of PD-L1 expression from the blood of WT mice. Monocyte subsets were defined as shown in fig. S1A, CMs were Ly6C+, IMs were Ly6Cint, and NCMs were Ly6C. Other leukocyte populations (CD45+) were gated as follows: B cells (CD11b, B220+), CD8 T cells (CD11b, B220, CD8+), CD4 T cells (CD11b, B220, CD4+), eosinophils (CD11b+, Ly6G, Sschigh), and neutrophils (PMNs, CD11b+, CD115, Ly6G+). Data are shown as means ± SEM. n = 4 mice from two independent experiments; ***P < 0.001. (C) Representative immunofluorescence confocal micrographs of whole blood stained for PD-L1 (cyan), CD115 (red), and Ly6G (yellow). Scale bar, 20 μm. (D) Flow cytometry–derived t-SNE reduction maps of whole-blood leukocytes. PD-L1 expression was compared with CX3CR1 expression from Cx3cr1gfp/+ mice. NCMs and CMs are indicated. (E) Frequency of blood PD-L1+ monocyte subsets. Representative flow cytometry analysis (left) and quantification (right) of PD-L1+ CMs, IMs, and NCMs from the blood of WT mice. Data are shown as means ± SEM. n = 4 mice from two independent experiments; ***P < 0.001. (F) Frequency of blood CX3CR1+ monocyte subsets. Representative flow cytometry analysis (left) and quantification (right) of CX3CR1+ CMs, IMs, and NCMs from the blood of Cx3cr1gfp/+ mice. Data are shown as means ± SEM. n = 3 mice. (G) Top: Representative flow cytometry analysis comparing gating strategies based on PD-L1 (left) and CX3CR1 (right) expression. Gates show all PD-L1+ and CX3CR1+ leukocytes. Bottom: Fraction of NCMs from the parental gates. AF, autofluorescence. (H) Percentage of PD-L1+ and CX3CR1+ leukocytes compared with the percentage of NCMs measured by conventional gating strategy (from fig. S1A). (I) Correlation between percentages of NCMs and PD-L1+ leukocytes. WBC, white blood cells. (J) Quantification of the NCMs fraction in PD-L1+ and CX3CR1+ leukocytes. Data in (H) and (J) are shown as means ± SEM. n = 4 mice (NCMs and PD-L1) and n = 3 mice (CX3CR1); **P < 0.01, ***P < 0.001. NS, not significant.

  • Fig. 2 In vivo tracking of PD-L1+NCMs in the microcirculation.

    (A) Representative 3D time series showing PD-L1+ NCMs (red) crawling in the vasculature of the cremaster muscle. Tracks are shown in white. Vessels were reconstructed on the basis of intrinsic autofluorescent signals (see also movie S1). Arrows show flow direction. Yellow inset is shown in detail in (C). Scale bars, 30 μm. (B) 3D track plot of PD-L1+ NCMs from a single 10-min recording showing flow-independent trajectories in all directions. (C) Details from the yellow inset in (A), showing representative cell tracks color-coded according to their length, duration, average speed, and straightness (confinement ratio, C.R.), as depicted. (D to F) Analysis of cell motility. (D) Distribution of PD-L1+ NCM confinement ratio and (E) comparison between measured (red line, best fitted curve; see Materials and Methods) and random (green dashed line, predicted by linear regression fitted on the earliest time intervals) mean cell displacement over time. Values are shown as means ± 95% confidence interval (red dotted lines). (F) Average speed. Lines represent means. All data refer to 195 cells pooled from three independent experiments.

  • Fig. 3 PD-L1 selectively distinguishes NCMs and IMs in the BM.

    (A) Representative flow cytometry analysis of PD-L1 and CX3CR1 expression on BM monocytes from WT and Cx3cr1gfp/+ mice. Monocytes subsets were gated as shown in Fig. 1A. (B) Quantitative analysis of PD-L1 and CX3CR1 expression on BM monocyte subsets. Data are shown as means ± SEM. n = 4 mice (WT) from two independent experiments and n = 3 mice (Cx3cr1gfp/+ mice); ***P < 0.001. (C) Difference of PD-L1 and CX3CR1 mean fluorescent intensity (MFI) between NCMs and CMs. Data are shown as means ± SEM. n = 4 mice (WT) from two independent experiments and n = 3 (Cx3cr1gfp/+ mice); **P < 0.01. (D) Representative 3D reconstruction of a region in the femoral BM stained for PD-L1 (blue), CD115 (red), and Gr-1 (green). White is bone collagen–derived SHG. Scale bars, 20 μm. (E) 3D segmentation from (D) according to fluorescent color combination (CD115 Gr-1high, PMNs in green; CD115+ Gr-1, NCMs in violet; CD115+ Gr-1low, IMs in pink; and CD115+ Gr-1+, CMs in red). Examples of segmented monocyte subsets are indicated by an arrowhead for CM, arrow for NCM, and asterisk for IM. (F) Representative 3D reconstructions of the BM in the femoral epiphysis and diaphysis stained for PD-L1 (yellow). Scale bars, 80 μm. (G) Absolute quantification of PD-L1+ cells in epiphysis (Epi) and diaphysis (Dia). Data are shown as means ± SEM. n = 4 mice. **P < 0.01.

  • Fig. 4 PD-L1+NCMs are enriched in BM regions with higher density of TZ vessels.

    (A) Representative 3D reconstructions of BM monocytes in the femoral epiphysis and diaphysis. Monocytes (CD115+) were classified after segmentation based on the expression of PD-L1 and Gr-1. CMs (red), IMs (pink), and NCMs (violet). Scale bars, 80 μm. (B) Distribution of monocyte subsets in epiphysis and diaphysis. Percentage (top) and absolute quantification (bottom) of monocyte subsets. Values are expressed as means (top) and means ± SEM (bottom). Data are representative of four independent experiments. *P < 0.05, ***P < 0.001. (C) Representative micrograph of the femoral vessel architecture in distal epiphysis and metaphysis. Whole-mounted femur was stained for laminin (green), and PDGFRβ1 (red) and stitched maximum intensity projection was generated (I). Inset is magnified on the right. (II) Merge and single channels are shown, as well as the 3D surface reconstruction of sinusoids (asterisk, laminin+ PDGFRβ1+), arterioles (arrow, lamininbright PDGFRβ1), and TZ vessels (arrowhead, laminin PDGFRβ1bright). Scale bar, 500 μm. (D) Correlation between the number of PD-L1+ monocytes (IMs and NCMs) and the surface of TZ vessels, sinusoids, and arterioles. Data were collected from 12 random areas of the femur deriving from four independent experiments. Spearman’s coefficients are indicated; *P < 0.05. fov, field of view. (E) Ratio between the TZ vessel surface and BM volume in epiphysis and diaphysis. Data are shown as means ± SEM. n = 6 fov from three mice. ***P < 0.001. (F) Percentage of CMs, IMs, and NCMs among monocytes in contact with TZ vessels in epiphysis and diaphysis. Data refer to 231 (epiphysis) and 137 (diaphysis) cells from two independent experiments. (G) Representative 3D reconstruction showing monocyte subsets and vessels in the epiphysis. CMs (red spheres), IMs (pink spheres), and NCMs (violet spheres). Sinusoids (yellow) and TZ vessels (green). Inset is magnified on the right. Scale bar, 80 μm. (H) Distance index of epiphyseal monocyte subsets. Left: Distance index of all monocytes to the closest sinusoid and TZ vessel. Right: Distance index of CMs, IMs, and NCMs closest to the TZ vessels. In the violin plots, center lines represent the median, and dots represent cells. n = 1955 monocytes (left); n = 470 CMs, n = 35 IMs, and n = 79 NCMs (right) from two independent experiments.

  • Fig. 5 Spatiotemporal analysis of monocyte conversion in the BM.

    (A to F) Conversion assessed by TPLSM imaging of the BM after adoptive transfer of CD45.1+ CMs into CD45.2 recipient. (A) Representative 3D reconstructions of CD45.1+ donor–derived monocytes (green) in epiphysis and diaphysis of CD45.2 recipient mice 24 hours after transfer of CMs. Scale bars, 80 μm. (B) Absolute quantification of CD45.1+ donor–derived monocytes in the epiphysis and diaphysis of CD45.2 recipient mice 24, 48, and 72 hours after transfer of CMs. Data are shown as means ± SEM. n = 2 mice per time point. *P < 0.05, **P < 0.01. (C) Percentage of CD45.1+ monocyte subsets in the epiphysis and diaphysis 24, 48, and 72 hours after adoptive transfer. n = 2 mice per time point. (D) Representative 3D reconstructions of epiphyseal regions showing the position of CMs (red spheres), IMs (pink spheres), and NCMs (violet spheres) in relation to sinusoids (yellow) and TZ vessels (green) 24, 48, and 72 hours after transfer. Scale bars, 80 μm. (E) Percentage of monocyte subsets among CD45.1+ monocytes in contact with TZ vessels at all time points after adoptive transfer. n = 2 mice per time point. (F) Distance index of CD45.1+ CMs, IMs, and NCMs to the closest sinusoid and TZ vessel at the indicated time points after transfer. In the violin plots, center lines represent the median, and dots represent cells. n = 656, 723, and 344 CMs, n = 246, 360, and 154 IMs, and n = 211, 145, and 271 NCMs are pooled from two mice per time point.

  • Fig. 6 Conversion of CMs into NCMs is defective in the BM of aged mice.

    (A) Representative flow cytometry analysis, (B) percentages, and (C) absolute counts of monocyte subpopulations in the blood of young and aged mice. Data are shown as means (B) and means ± SEM (C). n = 8 (young) and 7 (aged) mice from three independent experiments. ***P < 0.001. (D) Representative 3D reconstructions of epiphyseal regions showing CMs (red spheres), IMs (pink spheres), and NCMs (violet spheres) as well as TZ vessels (green) in young and aged mice. White insets are tilted and shown in detail on the left. Scale bars, 80 μm. (E) Percentages of monocyte subsets in the epiphysis of young and aged mice. Data are shown as means of four mice. (F) Absolute quantification of NCMs in epiphysis of young and aged mice. Data are shown as means ± SEM. n = 4 mice per group. **P < 0.01. (G) Absolute quantification of CMs in epiphysis of young and aged mice. Data are shown as means ± SEM. n = 4 mice per group. *P < 0.05. (H) Ratio between the TZ vessel surface and BM volume in epiphysis of young and aged mice. Data are shown as means ± SEM. n = 10 fov from four mice. ***P < 0.001.

  • Fig. 7 PD-L1 expression is maintained on NCMs under inflammatory conditions and PD-L1+NCMs are found in TLOs.

    (A) Representative flow cytometry and (B) quantification of PD-L1 expression on blood leukocytes in mice after sham or MI surgery. Data are shown as means ± SEM. n = 3 (sham) and n = 6 (MI) mice. ***P < 0.001. (C) Quantification of NCMs in PAT of mice after sham or MI surgery. Data are shown as means ± SEM. n = 5. ***P < 0.001. (D) Quantification of PD-L1 expression on leukocyte populations in PAT of mice after sham or MI surgery. Data are shown as means ± SEM. n = 3. ***P < 0.001. (E) Representative 3D TPLSM reconstruction of a TLO in PAT of mice showing clustered B cells (B220+, blue), T cells (CD3+, green), and monocytes (CD115+, red). Adipocyte-derived autofluorescence is depicted in yellow. Scale bar, 90 μm. (F) Detailed TPLSM micrography of PD-L1+ (white), CD115+ (red) NCMs (indicated by the arrows), B220+ (blue) B cells, and CD3+ (green) T cells in a TLO in PAT of mice. Single markers are shown on the right. Adipocyte-derived autofluorescence is depicted in yellow. Scale bar, 20 μm. (G) Representative 3D TPLSM reconstruction of CD115+ PD-L1+ NCMs (yellow spheres) and CD31+ blood vessels (blue) in a TLO of PAT. Three examples of NCMs in contact with the extraluminal side of the vascular wall (tilted insets) are shown in detail. Scale bar, 30 μm. (H) Confocal laser scanning microscopy (CLSM) image of a TLO in human adventitial fat of the aorta, showing CD20+ (red) B cells and PD-L1+ (white), CD14+ (green), CD16+ (blue) NCMs. Representative PD-L1+ NCMs are indicated by Roman numerals and shown in detail and as single-color micrographs on the right. Scale bars, 20 μm and 5 μm.

  • Fig. 8 NCMs regulate T cell survival in TLOs via PD-L1.

    (A to C) Increase of NCM–T cell contacts in TLOs in the PAT of mice 3 days after MI. (A) Representative TPLSM 3D reconstruction of PD-L1+ NCMs (red spheres) and T cells (cyan spheres) after MI (right) or sham (left) surgery. Examples of NCM–T cell contacts are indicated by the arrows. Scale bars, 30 μm. (B) TPLSM-derived quantification of PD-L1+ NCMs normalized on volume of TLOs. Data are shown as means ± SEM. n = 8 (sham) and n = 10 (MI) TLOs from three to five mice, respectively. **P < 0.01. (C) Percentage of PD-L1+ NCMs in direct contact with T cells in single TLOs. Data are shown as mean ± SEM. n = 9 (sham) and n = 10 (MI) TLOs from three to five mice, respectively. ***P < 0.001 (D) Representative single-plane TPLSM micrograph of a PD-L1+ (white) CD115+ (red) NCM in contact with CD3+ (green) T cells and B220+ (blue) B cells. PD-L1 at the NCM–T cell interface is indicated by the arrow. Scale bar, 10 μm. (E to J) Effect of treatment with control IgG or the anti–PD-L1 antibody on NCMs and T cells in TLOs of mice 3 days after MI. (E) Representative TPLSM reconstructions of CD115+ Ly6C NCMs (red spheres) and T cell (cyan spheres). NCMs in direct contact with T cells are depicted as yellow spheres. Insets are shown in detail at the bottom. Scale bars, 80 μm. (F) Percentage of CD115+ Ly6C NCMs in direct contact with T cells. Data are shown as means ± SEM. n = 7 (sham) and n = 9 (MI) TLOs from three mice per group. *P < 0.05. (G) Absolute quantification of CD115+ Ly6C NCMs per volume of TLOs. Data are shown as means ± SEM. n = 7 (sham) and n = 9 (MI) TLOs from three mice per group. (H) Absolute quantification of T cells in single TLOs in the PAT of mice 3 days (3d; left) and 5 days (right) after MI and treatment. Data are shown as means ± SEM. n > 11 TLOs from three to four mice per group. *P < 0.05, **P < 0.01. (I) Representative TPLSM reconstructions of Annexin V+ (pink spheres) and Annexin V (cyan spheres) T cells after treatment. Insets are shown in detail and as single colors at the bottom. Scale bars, 80 μm. (J) Percentage of Annexin V+ T cells in single TLOs 3 days after MI. Data are shown as means ± SEM. n > 11 TLOs from three to four mice per group. ***P < 0.001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/36/eaar3054/DC1

    Materials and Methods

    Fig. S1. In silico identification of candidate markers for BM and PB NCMs.

    Fig. S2. Anti–PD-L1 antibody is preferable to Cx3cr1gfp/+ mice to mark circulating NCMs.

    Fig. S3. Anti-CD43 antibody does not specifically mark circulating NCMs.

    Fig. S4. PD-L1 expression is strongly reduced in Nr4a1−/− mice lacking NCMs.

    Fig. S5. Kinetics and concentration of the anti–PD-L1 antibody for in vivo labeling of NCMs.

    Fig. S6. CD43 does not specifically distinguish NCMs in the BM.

    Fig. S7. CMs are the main monocyte subset in physical contact with TZ vessels in the diaphysis, whereas NCMs are in proximity of sinusoids.

    Fig. S8. Transfer of purified CMs and BrdU pulse-chase experiment.

    Fig. S9. PD-L1 expression is maintained on NCMs under inflammatory conditions, and PD-L1+ NCMs are found in TLOs.

    Fig. S10. Anti–PD-L1 treatment does not deplete circulating NCMs.

    Table S1. Association between the number of PD-L1+ cells and vessel subtypes.

    Table S2. List of antibodies.

    Movie S1. In vivo tracking of patrolling PD-L1+ NCMs in the cremaster microcirculation.

    Data file S1. List of genes differentially expressed in CMs and NCMs.

    Data file S2. Raw data from main figures.

    Data file S3. Raw data from supplementary figures.

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. In silico identification of candidate markers for BM and PB NCMs.
    • Fig. S2. Anti–PD-L1 antibody is preferable to Cx3cr1gfp/+ mice to mark circulating NCMs.
    • Fig. S3. Anti-CD43 antibody does not specifically mark circulating NCMs.
    • Fig. S4. PD-L1 expression is strongly reduced in Nr4a1−/− mice lacking NCMs.
    • Fig. S5. Kinetics and concentration of the anti–PD-L1 antibody for in vivo labeling of NCMs.
    • Fig. S6. CD43 does not specifically distinguish NCMs in the BM.
    • Fig. S7. CMs are the main monocyte subset in physical contact with TZ vessels in the diaphysis, whereas NCMs are in proximity of sinusoids.
    • Fig. S8. Transfer of purified CMs and BrdU pulse-chase experiment.
    • Fig. S9. PD-L1 expression is maintained on NCMs under inflammatory conditions, and PD-L1+ NCMs are found in TLOs.
    • Fig. S10. Anti–PD-L1 treatment does not deplete circulating NCMs.
    • Table S1. Association between the number of PD-L1+ cells and vessel subtypes.
    • Table S2. List of antibodies.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). In vivo tracking of patrolling PD-L1+ NCMs in the cremaster microcirculation.
    • Data file S1 (Microsoft Excel format). List of genes differentially expressed in CMs and NCMs.
    • Data file S2 (Microsoft Excel format). Raw data from main figures.
    • Data file S3 (Microsoft Excel format). Raw data from supplementary figures.

    Files in this Data Supplement:

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