Research ArticleGraft-Versus-Host Disease

Graft-versus-host disease, but not graft-versus-leukemia immunity, is mediated by GM-CSF–licensed myeloid cells

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Science Translational Medicine  28 Nov 2018:
Vol. 10, Issue 469, eaat8410
DOI: 10.1126/scitranslmed.aat8410
  • Fig. 1 Donor T cells secrete GM-CSF and IFN-γ during allogeneic responses.

    (A) Survival of lethally irradiated CD45.2+ BALB/c mice after allo-HCT with CD45.1+ WT C57BL/6 TCD-BM alone or combined with 10 × 106 CD45.1+ WT C57BL/6 splenocytes. Data were pooled from four individual experiments, each with n = 5 per group. (B) Frequency of CD45.1+ cells within live singlets in the spleen, liver, and skin at 3 and 6 days after allo-HCT. Data were pooled from two experiments to obtain n = 8 to 10 per group. (C) Frequency of IFN-γ–, GM-CSF–, and IL-17A–producing CD4+ and CD8+ T cells within the CD45.1+ population from spleens of mice 3 days after allo-HCT. Representative plots are shown from a total of three independent experiments. (D) Frequencies of IFN-γ–, GM-CSF–, and IL-17A–producing CD4+ and CD8+ T cells within the CD45.1+ populations from liver, spleen, and skin 3 and 6 days after allo-HCT. Data were pooled from three individual experiments, total n = 8 to 10 per group. (E) Frequencies of IFN-γ– and GM-CSF–producing CD4+ and CD8+ T cells within the CD45.1+ populations from liver and spleen 3 and 6 days after allo-HCT. Representative data from one of three experiment are shown, each with n = 3 to 4 per group. (F) Serum IFN-γ and GM-CSF in mice 6 days after allo-HCT. Data were pooled from three individual experiments, each with n = 2 to 5 per group. (G) IFN-γ, GM-CSF, and IL-17A in supernatants from cocultures of T cells from WT C57BL/6 mice with either syngeneic (C57BL/6) or allogeneic (BALB/c) splenic CD11c+ DCs. Data were pooled from three individual experiments, each with n = 3 to 5 per group. (H) Tritiated thymidine incorporation by T cells from C57BL/6 WT, Ifng−/−, Csf2−/−, or Il17a/− mice cocultured with syngeneic (C57BL/6) or allogeneic (BALB/c) splenic CD11c+ DCs. Data were pooled from three individual experiments, each with n = 2 to 5 per group. For comparison of survival curves, a log-rank (Mantel-Cox) test was used in (A). For comparison of the means, an unpaired two-tailed t test with Welch’s correction was used in (B), (F), and (G), and one-way analysis of variance (ANOVA) with Bonferroni posttest was used in (D) and (H). *P < 0.05, **P < 0.01, ***P < 0.001. Data are shown as means ± SEM. cpm, counts per minute; ns, not significant.

  • Fig. 2 GM-CSF is crucial for acute GvHD after fully MHC-mismatched allo-HCT.

    (A) Survival of lethally irradiated BALB/c mice after allo-HCT with WT C57BL/6 TCD-BM alone or with 10 × 106 splenocytes from C57BL/6 WT Csf2−/−, Ifng−/−, or Il17a−/− mice. Data were pooled from four individual experiments, each with n = 5 per group. (B) Survival of lethally irradiated BALB/c mice after allo-HCT with WT C57BL/6 TCD-BM alone or combined with 3 × 106 T cells purified from spleens of C57BL/6 WT or Csf2−/− mice. Data were pooled from three individual experiments, each with n = 5 per group. (C) Composite histopathological score for liver, small intestine, and skin from BALB/c mice 6 days after allo-HCT with WT C57BL/6 TCD-BM combined with 10 × 106 splenocytes from either WT or Csf2−/− C57BL/6 mice. Data were pooled from three individual experiments, each with n = 5 per group. (D) Representative images of hematoxylin and eosin–stained sections from skin and small intestine of mice 6 days after allo-HCT described in (A), each with n = 4 to 5 per group. Scale bars, 100 μm. (E) Colon length in centimeters from BALB/c mice 6 days after allo-HCT, as described in (C). Data were pooled from two individual experiments. (F) Representative images and quantification of apoptotic cells [TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) staining] in the colon from BALB/c mice 6 days after allo-HCT, as described in (C). Data were pooled from three individual experiments. Scale bars, 50 μm (top) and 20 μm (bottom). (G) Concentrations of AP, ALT, BUN, and albumin in serum from BALB/c mice 6 days after allo-HCT, as described in (C). Data are representative of two individual experiments, each with n = 3 to 5 per group. (H) Representative images of p22phox labeling in sections from liver, small intestine, and skin of mice 6 days after allo-HCT, as described in (C) (left). Scale bars, 100 μm. Data were pooled from four independent experiments, each with n = 5 to 7 per group. Quantification of mean percentage of total area labeled positively for p22phox per visual field (right). (I) Survival of lethally irradiated BALB/c mice after allo-HCT with WT C57BL/6 TCD-BM alone or combined with 10 × 106 splenocytes from C57BL/6 WT mice. Mice were treated with phosphate-buffered saline (PBS), 300 μg of isotype control antibody, or 300 μg of anti–GM-CSF antibody (a–GM-CSF) injected intraperioneally three times per week for the duration of the experiment, starting 2 days before HCT. Data were pooled from two individual experiments, each with n = 5 per group. For comparison of survival curves, a log-rank (Mantel-Cox) test was used in (A) (WT versus other groups), (B) (WT to Csf2−/−), and (I) (a–GM-CSF versus isotype). For comparison of the means (WT versus Csf2−/−), an unpaired two-tailed t test was used in (C), (E), (F), and (H). *P < 0.05, **P < 0.01, ***P < 0.001. Data are shown as means ± SEM.

  • Fig. 3 GM-CSF mediates GvHD pathology after partially MHC-mismatched allo-HCT.

    (A) Survival of lethally irradiated B6D2F1 mice after partially MHC-mismatched allo-HCT with WT C57BL/6 TCD-BM alone or combined with 20 × 106 splenocytes from C57BL/6 WT, Csf2−/−, or Ifng−/− mice. Data were pooled from five individual experiments, each with n = 5 per group. (B) Survival of lethally irradiated B6D2F1 mice after allo-HCT with WT C57BL/6 TCD-BM alone or combined with 7.5 × 106 T cells purified from spleens of C57BL/6 WT, Csf2−/−, or Ifng−/− mice. Data were pooled from two individual experiments, each with n = 5 per group. (C) Composite histopathological score for liver, small intestine, and skin sections from B6D2F1 mice at 11 days after allo-HCT, described in (A). Data were pooled from two individual experiments, each with n = 5 per group. (D) Representative images of hematoxylin and eosin–stained sections from skin of mice 11 days after allo-HCT with WT C57BL/6 TCD-BM combined with 20 × 106 splenocytes from either WT, Ifng−/−, or Csf2−/− C57BL/6 mice, each with n = 4 to 5 per group. Scale bars, 100 μm. (E) Composite histopathological score for liver sections from B6D2F1 mice at 9 or 28 days after allo-HCT, as described in (A). Data are representative of two independent experiments, each with n = 4 to 6 per group. (F and G) Representative images of p22phox (top panels) and F4/80 labeling in sections from the (F) liver and (G) small intestine of mice 9 days after allo-HCT with WT C57BL/6 TCD-BM combined with 20 × 106 splenocytes from C57BL/6 WT or Csf2−/− mice. Scale bars, 100 μm. Data were pooled from four independent experiments, each with n = 5 to 7 per group. Quantification of percentage of total area labeled positively for p22phox or F4/80 per visual field. (H) Serum IFN-γ and GM-CSF in mice 9 days after allo-HCT with WT C57BL/6 TCD-BM combined with 20 × 106 splenocytes from C57BL/6 WT, Ifng−/−, or Csf2−/− mice. Data were pooled from two to three individual experiments, each with n = 4 to 5 per group. For comparison of survival curves (WT versus other groups), a log-rank (Mantel-Cox) test was used in (A) and (B). For comparison of the means (WT, Csf2−/−, and Ifng−/−), one-way ANOVA with Bonferroni posttest was used in (C). For comparison of the means (WT versus Csf2−/−), an unpaired two-tailed t test was used in (F) and (G). For comparison of the means (WT versus Ifng−/− or WT versus Csf2−/−), an unpaired two-tailed t test with Welch’s correction was used in (H). *P < 0.05, **P < 0.01, ***P < 0.001. Data are shown as means ± SEM.

  • Fig. 4 GM-CSF drives GvHD through donor-derived myeloid cells.

    (A) Survival of lethally irradiated WT C57BL/6 and Csf2rb−/− mice after MHC-mismatched allo-HCT with WT BALB/c TCD-BM alone or combined with 20 × 106 splenocytes from BALB/c WT mice. Data were pooled from two individual experiments, each with n = 5 per group. (B) Survival of lethally irradiated BALB/c WT mice following MHC-mismatched allo-HCT with C57BL/6 WT or and Csf2rb−/− mice TCD-BM alone or combined with 10 × 106 splenocytes from C57BL/6 WT mice. Data were pooled from two individual experiments, each with n = 5 per group. (C) Annotated t-SNE map displaying 200,000 randomly sampled cells from the BM of WT C57BL/6 and Csf2rb−/− mice showing STAT5 phosphorylation (pSTAT5; black to yellow gradient) upon GM-CSF stimulation, analyzed by flow cytometric analysis. Data represent two independent experiments, each with n = 3 per group. (D) Frequencies of GM-CSF–induced pSTAT5 up-regulation in monocytes and neutrophils from WT C57BL/6 and Csf2rb−/− mice as shown overlaid in (B). (E) Flow cytometric analysis of different myeloid cell populations (DCs, neutrophils, and monocytes and MDCs) after HCT. Example gating for the liver is shown. (F) Quantification of myeloid cell populations [from (E)] in the spleen (top row) and liver (bottom row). One representative from three individual experiments is shown, each with n = 4 to 5 per group. (G) Frequency of pro–IL-1β–producing neutrophils and monocytes within the H2Db+ CD45+ population from spleens of mice 6 days after allo-HCT. Representative plots are shown for neutrophils, monocytes, and MDCs [gated as in (E)]. Data represent two independent experiments, each with n = 5 per group. (H) Flow cytometric analysis of ROS (CellROX reagent) in mice 6 days after allo-HCT. Representative histograms of median fluorescence intensity (MFI) for neutrophils, monocytes, and MDCs [gated as in (E)]. Data represent one experiment with n = 5 to 7 per group. For comparison of survival curves (WT versus Csf2rb−/− recipients/donors), a log-rank (Mantel-Cox) test was used in (A) and (B). For comparison of the means, an unpaired two-tailed t test was used in (D) and (F) to (H). ***P < 0.001, **P < 0.01, *P < 0.05. Data are shown as means ± SEM.

  • Fig. 5 GM-CSF is dispensable for antitumor activity after allo-HCT.

    (A to D) Lethally irradiated BALB/c mice were intravenously injected with 250,000 A20 tumor cells expressing GFP and luciferase at the same time as MHC-mismatched allo-HCT with WT C57BL/6 TCD-BM alone or combined with 1 × 105 T cells purified from spleens of C57BL/6 WT or Csf2−/− mice. Mice treated with TCD-BM alone were used as controls. (A) Tumor growth was monitored by in vivo BLI. Images from one representative experiment of three are shown. (B) Signal intensity in the region of interest (ROI) was monitored over time. (C) Survival over time and (D) Incidence of GvHD shown as percentage of mice in each treatment group which developed lethal GvHD over time (gray) or survivors (white). Data were pooled from three individual experiments, each with n = 5 per group. (E to H) Lethally irradiated BALB/c mice were intravenously injected with A20 tumor cells expressing GFP and luciferase at the same time as MHC-mismatched allo-HCT with WT C57BL/6 TCD-BM alone or combined with 1 × 105 T cells purified from spleens of C57BL/6 WT mice. Mice treated with TCD-BM alone were used as controls. Mice were treated with isotype control or anti–GM-CSF antibody three times per week for the duration of the experiment, starting 2 days before HCT. (E) Tumor growth was monitored by in vivo BLI. Images from one experiment with n = 6 to 7 per group are shown. (F) Signal intensity in the region of interest was monitored over time. (G) Survival over time and (H) incidence of GvHD shown as percentage of mice in each treatment group which developed lethal GvHD over time (gray) or survivors (white). For comparison of survival curves [WT versus other groups in (C) and WT versus csf2−/− in (G)], a log-rank (Mantel-Cox) test was used. *P < 0.05 and **P < 0.01. Data are shown as means ± SEM.

  • Fig. 6 GM-CSF+ T cells are elevated in patients with GvHD.

    (A) Relative expression of GM-CSF at the mRNA level in gastrointestinal biopsies from patients with different GvHD grades (see table S1). Data are shown as means ± SEM. (B) Images of GM-CSF labeling in control (patients with no pathological findings) and GvHD grade IV biopsies from the small intestine of allo-HCT patients (see tables S2 and S3). Brown, anti-human GM-CSF; blue, hematoxylin. Scale bars, 100 and 20 μm in zoom images. Representative images of three individual control and patient samples are shown (tables S2 and S3). (C) Immunofluorescence staining for CD3 (pink), CD68 (green), and GM-CSF (red) of gastrointestinal biopsies from patients with grade IV GvHD (table S2). A representative picture of three individual patient samples is shown. Nuclei are depicted in blue (4ʹ,6-diamidino-2-phenylindole). Scale bars, 50 μm (top) and 20 μm (bottom). (D) Flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) collected from healthy donors (HD) and patients with GvHD (GP) (table S4), stimulated for 4 hours with phorbol 12-myristate 13-acetate/ionomycin. Left: Representative plots of cytokine expressing T cells are shown. Right: GM-CSF–, IFN-γ–, and IL-17–producing CD4+ and CD8+ T cells are presented as individual frequencies of CD4 or CD8 T cells, n = 9 to 10 per group. For comparison of the means, one-way ANOVA with Bonferroni posttest was used in (A), and an unpaired two-tailed Mann-Whitney test was used in (D). *P < 0.05 and **P < 0.01.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/469/eaat8410/DC1

    Materials and Methods

    Fig. S1. Comparative phenotypic analysis of T cell populations from WT and Csf2−/− mice.

    Fig. S2. GM-CSF is crucial for acute GvHD after fully MHC-mismatched allo-HCT.

    Fig. S3. GM-CSF mediates GvHD pathology after partially MHC-mismatched allo-HCT.

    Fig. S4. GM-CSF drives GvHD through donor-derived myeloid cells.

    Fig. S5. GM-CSF is dispensable for antitumor activity after allo-HCT.

    Fig. S6. Frequency of cytokine-expressing T cells in PBMCs of patients with GvHD.

    Table S1. Clinicopathological features of GvHD patient set I.

    Table S2. Clinicopathological features of GvHD patient set II.

    Table S3. Characteristics of control subjects.

    Table S4. Clinicopathological features of GvHD patient PBMCs.

    Table S5. Primary data.

    Table S6. GvHD scoring system for allo-HCT mice.

    Table S7. Phenotypic analysis of human PBMCs by flow cytometry.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Comparative phenotypic analysis of T cell populations from WT and Csf2−/− mice.
    • Fig. S2. GM-CSF is crucial for acute GvHD after fully MHC-mismatched allo-HCT.
    • Fig. S3. GM-CSF mediates GvHD pathology after partially MHC-mismatched allo-HCT.
    • Fig. S4. GM-CSF drives GvHD through donor-derived myeloid cells.
    • Fig. S5. GM-CSF is dispensable for antitumor activity after allo-HCT.
    • Fig. S6. Frequency of cytokine-expressing T cells in PBMCs of patients with GvHD.
    • Table S1. Clinicopathological features of GvHD patient set I.
    • Table S2. Clinicopathological features of GvHD patient set II.
    • Table S3. Characteristics of control subjects.
    • Table S4. Clinicopathological features of GvHD patient PBMCs.
    • Legend for table S5
    • Table S6. GvHD scoring system for allo-HCT mice.
    • Table S7. Phenotypic analysis of human PBMCs by flow cytometry.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S5 (Microsoft Excel format). Primary data.

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