Research ArticleAutoimmunity

PD-1H (VISTA)–mediated suppression of autoimmunity in systemic and cutaneous lupus erythematosus

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Science Translational Medicine  11 Dec 2019:
Vol. 11, Issue 522, eaax1159
DOI: 10.1126/scitranslmed.aax1159
  • Fig. 1 PD-1H KO mice on a BALB/c background develop spontaneous systemic and cutaneous autoimmunity resembling lupus.

    (A) The appearance of representative female WT BALB/c mice and female BALB/c PD-1H KO mice with spontaneous periorbital and dorsal nape dermatitis. (B) Incidence of spontaneous cutaneous lupus in female WT mice (n = 62) and female PD-1H KO mice (n = 66) over time. (C) Representative histological images of healthy skin from WT mice and affected skin from PD-1H KO mice, MRL/lpr mice, and human DLE (original magnification, ×400) stained with hematoxylin and eosin (H&E). Scale bars, 20 μm. (D) Skin biopsy score based on inflammation, acanthosis, and ulceration in female WT (n = 20) and PD-1H KO mice (n = 20). (E) Representative images of spontaneous pericardial calcification observed in 10-month-old PD-1H KO mice in comparison with 10-month-old WT mice. H&E of the pericardium from WT and PD-1H KO mice. Von Kossa insert (original magnification ×100 for histology). Scale bar, 100 μm. (F) Quantification of pericardial calcification as evaluated by the percentage of the area of the heart covered by calcium for 5-month-old female WT (n = 15) and PD-1H KO mice (n = 20) and 10-month-old female WT (n = 16) and PD-1H KO mice (n = 18) (0, none; 1, <10%; 2, 10 to 30%; 3, >30%). (G) Serum concentrations of ANA from 3-month-old female WT (n = 11) and PD-1H KO mice (n = 12) and 12-month-old female WT (n = 5) and PD-1H KO mice (n = 8). (H) Serum concentrations of anti-dsDNA IgG from 6-month-old female WT (n = 19) and PD-1H KO mice (n = 21). PD-1H KO mice are subdivided into those mice with cutaneous lupus lesions (lesion+; n = 11) and without cutaneous lupus lesions (lesion−; n = 10). (I) Concentrations of IL-6 and MCP-1 in sera of 9-month-old female WT (n = 15) and PD-1H KO mice (n = 29). Log-rank (Mantel-Cox) shown in (B). Bar graphs with mean ± SEM are shown in (D) and (F), and box and whisker plots are shown in (G), (H), and (I) analyzed with unpaired Student’s t test. *P < 0.05, **P < 0.01, ****P < 0.0001; ns, nonsignificant.

  • Fig. 2 Global immune overactivation of PD-1H KO mice.

    (A) Total cell counts of lymph nodes (LN; pooled cell suspension of bilateral axillary, bronchial, and inguinal lymph nodes) and spleens (SPL) of female 5-month-old WT (n = 10) and PD-1H KO mice (n = 11). (B) Total number of CD3+ T cells, CD4+ T cells, CD8+ T cells, NKs, CD11b+ cells, and pDCs (CD11c+B220+PDCA-1+) in pooled lymph nodes of female 5-month-old WT (n = 6) and PD-1H KO mice (n = 7). (C) Percentages of TFH cells (CD3+CD4+CXCR5+PD-1+Bcl6+) in lymph nodes and spleens of female 5-month-old WT (n = 2) and PD-1H KO mice (n = 4). (D) Effector memory T cell percentages in lymph nodes and spleen of female 5-month-old WT (n = 15) and PD-1H KO mice (n = 13). (E) Percentages of Tregs in lymph nodes and spleen of female 5-month-old WT (n = 15) and PD-1H KO mice (n = 13). Percentages of Tregs are also compared between PD-1H KO mice with cutaneous lupus (lesion+) (n = 5) and without cutaneous lupus (lesion−; n = 8). Data are presented as box and whisker plots of measurements obtained in duplicate or triplicate experiments. *P < 0.05, **P < 0.01 by unpaired Student’s t test or Mann-Whitney U test for nonnormalized data as determined by Kolmogorov-Smirnov test for normality for CD11b+ cells in (B). ns, nonsignificant.

  • Fig. 3 Neutrophils infiltrate cutaneous lupus lesions of PD-1H KO mice and are proinflammatory.

    (A) Representative images of immunohistochemistry (IHC) for CD3 (T cells), CD11b (myeloid cells), and CD317 (pDCs) from cutaneous lupus lesions of female PD-1H KO mice and cutaneous lupus lesions of MRL/lpr mice compared with age- and gender-matched WT controls. Bottom panel showing representative images of IHC for CD3 (T cells), CD68 (macrophages), and CD123 (pDC) from human DLE skin (magnification, ×400). Scale bars, 20 μm. (B) Quantification of IHC staining for CD3 and CD11b (top) and CD317 (bottom) in WT mice (n = 5) and lesional skin from MRL/lpr (n = 9) and PD-1H KO mice (n = 5) [hpf (high-power field), ×400]. (C) Representative viSNE graphs from mass cytometry analysis of healthy-appearing skin (single eyelid skin) from 6-month-old female WT or PD-1H KO mice as compared with established cutaneous lupus from female PD-1H KO mice (periorbital eyelid skin). (D) Quantification of mass cytometry data showing neutrophil percentages of infiltrated total CD45+ cells infiltrating periorbital (eyelid) skin from female 4-month-old WT (n = 3) and PD-1H KO mice (n = 3) and from female 6-month-old WT (n = 5) and PD-1H KO mice (n = 8). PD-1H KO mice are subdivided into those mice with cutaneous lupus lesions (lesion+; n = 4) and without cutaneous lupus lesions (lesion−; n = 4) in the third column (labeled as KO). (E) Neutrophils were enriched from bone marrow and pooled from three different female WT or PD-1H KO mice and stimulated by LPS (100 ng/ml) or not (no. sti). ELISA for IL-6, TNFα, and MCP-1 concentrations in culture medium are shown. Box and whisker plots are shown and analyzed by one-way ANOVA with Tukey post hoc analysis (B and D). Mean ± SEM shown in (E) and analyzed by unpaired Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 PD-1H KO mice develop more severe pristane-induced inflammatory arthritis and lupus.

    (A) Incidence of arthritis in female PD-1H KO (n = 12) and WT mice (n = 10) treated with pristane and representative of two independent experiments. Log rank (Mantel-Cox) analysis is shown. (B) Representative images of clinical appearance of front and hind legs from WT and PD-1H KO mice 6 months after pristane administration. (C) Representative images of H&E-stained hind limb ankle joint from WT and PD-1H KO mice 6 months after pristane administration. (D) Clinical arthritis score from female PD-1H KO (n = 12) and WT mice (n = 10) treated with pristane as determined by extent of joint swelling and inflammation. (E) Quantification of peritoneal neutrophils isolated from WT and PD-1H KO mice without treatment (WT, n = 3; KO, n = 3) or 4 days after pristane administration (WT, n = 6; KO, n = 5). (F) Serum concentrations of ANA and anti-histone Igs from female WT (n = 10) or PD-1H KO (n = 11) mice 6 months after pristane administration. Box and whisker plots are shown (D to F) with *P < 0.05 and ***P < 0.001 by unpaired Student’s t test.

  • Fig. 5 Up-regulation of PD-1H in patients with SLE and DLE.

    (A) Representative images of H&E and IHC of CD3, CD4, CD8, CD123 (pDCs), and PD-1H on DLE. (B) Quantification of IHC staining from DLE (n = 21), lichen planus (LP) (n = 11), and control skin (n = 20) FFPE tissue as displayed by average number of positive cells per high-powered field (×400). (C) Quantification of PD-1H expression within infiltrating CD3+ T cells, CD8+ T cells, CD68+ macrophages, and CD11b+ myeloid cells in DLE (n = 10 for CD3 and n = 6 for all others) and control (ctrl) skin (n = 10 for CD3 and n = 5 for all others) by mQIF using AQUA analysis algorithm. (D) Violin plot of human PD-1H mRNA expression (PD1H) as determined by fragments per kilobase millions (FPKM) of circulating immune cells (whole blood) from patients with SLE (n = 111) and healthy controls (n = 25). Box and whisker plots are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired Student’s t test or one-way ANOVA with Tukey post-hoc analysis (B). ns, nonsignificant.

  • Fig. 6 A PD-1H agonist reduces autoimmune lupus in MRL/lpr mice.

    (A) Incidence of cutaneous lupus in female MRL/lpr mice treated weekly with either 200 μg of control hamster IgG (n = 10) or agonist PD-1H MH5A (n = 10) from 6 to 16 weeks of age. Data are representative to two independent experiments. Log rank (Mantel-Cox) analysis. Subsequent panels displaying analysis of mice used in (A). (B) Representative clinical images of MRL/lpr mice treated with either MH5A or control IgG. (C) Quantification of cutaneous lupus severity based on the clinical scores of erythema, alopecia, erosions/crust, and lesion size of MRL/lpr mice treated with either MH5A or control IgG at 20 weeks. (D) Quantification of skin biopsy score based on histological analysis of inflammation, acanthosis, and ulceration from skin of MRL/lpr mice treated with either MH5A or control IgG. (E) Quantification of IHC of CD3, CD11b, and CD317 of skin from MRL/lpr mice treated with either MH5A or control IgG at 20 weeks. (F) ANA, ds-DNA (week 20), and anti-Ro52 (week 20) autoantibodies detected in sera of MRL/lpr mice treated with either MH5A or control IgG. (G) Quantification of T cells from lymph nodes of MRL/lpr mice treated with either MH5A or control IgG at 20 weeks as determined by multiparameter flow cytometry. (H) Quantification of pDCs (CD11c+CD317+), neutrophils (CD11b+Ly6C+Ly6G+), and TFH (CD4+CXCR5+PDhi) cells from lymph nodes of MRL/lpr mice treated with either MH5A or control IgG at 20 weeks as determined by flow cytometry. (I) Proinflammatory cytokines/chemokines detected in sera of MRL/lpr mice treated with either MH5A or control IgG at week 20 using cytokine multiplex array. Mean ± SEM is shown (C and D) or box and whisker plots (E to I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired Student’s t test.

  • Fig. 7 PD-1H stimulation attenuates T cell signaling and myeloid cell function.

    (A) Splenocytes from WT (red) and PD-1H-KO (gray) BALB/c mice were stained with PD-1H mAb or isotype control (open histogram) and cell surface markers. PD-1H expression in T cell (CD3+), pDC (B220+PDCA1+), myeloid cell (CD11b+), and B cell (B220+PDCA1) gates are shown. (B) Lymph nodes were isolated and pooled from three different female WT mice and stimulated with immobilized anti-CD3 (3 or 10 μg/ml) and PD-1H agonist MH5A (10 μg/ml) or IgG control (10 μg/ml). Thirty minutes after stimulation, cells were fixed, and phospho-flow cytometry was performed (percentages of phospho-Erk1/2+ CD4+ T cells are shown). (C) Purified pDCs from female WT (n = 3) or MRL/lpr mice (n = 3) were incubated with immobilized agonist PD-1H mAb MH5A or control IgG (10 μg/ml) and stimulated with R848 (50 μg/ml), and ELISA of culture media for IFN-α was performed. (D and E) Purified neutrophils from WT mice (n = 3 to 6) (D) or MRL/lpr mice (n = 3 to 6) (E) were incubated with immobilized agonist PD-1H mAb MH5A or control IgG (10 μg/ml) and stimulated with LPS (200 μg/ml), and cytokine array of culture media for IL-1β, TNFα, and MCP-1 was performed. (F) The granulocyte, monocyte, and macrophage numbers in the peritoneal cavity of WT BALB/c mice 4 days after pristane treatment. Mice were given two doses of 200 μg of either control IgG (n = 3) or anti–PD-1H MH5A mAb (n = 3) on days 0 and 2 after pristane treatment. Mean ± SEM is shown. *P < 0.05, **P < 0.01 by unpaired Student’s t test.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/522/eaax1159/DC1

    Fig. S1. PD-1H KO mice do not develop lupus nephritis.

    Fig. S2. Serum cytokines from WT and PD-1H KO mice.

    Fig. S3. PD-1H modulates type I IFNs, and type I IFN response genes are modestly up-regulated in cutaneous lupus lesions of PD-1H KO mice.

    Fig. S4. Quantification of mass cytometry from WT and PD-1H KO skin.

    Fig. S5. PD-1H KO mice and WT mice develop similar pristane-induced kidney disease.

    Fig. S6. IHC of human control skin, discoid lupus, and LP.

    Fig. S7. Multiplex immunofluorescence of human DLE and control skin.

    Fig. S8. Histology and immunophenotyping of MRL/lpr mice treated with a PD-1H agonist.

    Fig. S9. Proteinuria, kidney inflammation, and lupus nephritis in MRL/lpr mice treated with a PD-1H agonist.

    Fig. S10. The PD-1H agonist MH5A does not affect T cell signaling or pDC and neutrophil function from PD-1H KO mice.

    Table S1. Histological scoring of cutaneous lupus lesions.

    Table S2. Murine mass cytometry antibody panel.

    Data file S1. Primary data.

  • The PDF file includes:

    • Fig. S1. PD-1H KO mice do not develop lupus nephritis.
    • Fig. S2. Serum cytokines from WT and PD-1H KO mice.
    • Fig. S3. PD-1H modulates type I IFNs, and type I IFN response genes are modestly up-regulated in cutaneous lupus lesions of PD-1H KO mice.
    • Fig. S4. Quantification of mass cytometry from WT and PD-1H KO skin.
    • Fig. S5. PD-1H KO mice and WT mice develop similar pristane-induced kidney disease.
    • Fig. S6. IHC of human control skin, discoid lupus, and LP.
    • Fig. S7. Multiplex immunofluorescence of human DLE and control skin.
    • Fig. S8. Histology and immunophenotyping of MRL/lpr mice treated with a PD-1H agonist.
    • Fig. S9. Proteinuria, kidney inflammation, and lupus nephritis in MRL/lpr mice treated with a PD-1H agonist.
    • Fig. S10. The PD-1H agonist MH5A does not affect T cell signaling or pDC and neutrophil function from PD-1H KO mice.
    • Legends for tables S1 and S2
    • Legend for data file S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Histological scoring of cutaneous lupus lesions.
    • Table S2 (Microsoft Excel format). Murine mass cytometry antibody panel.
    • Data file S1 (Microsoft Excel format). Primary data.

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