Fig 2: BMP9 attenuates EPO/SCF-mediated expansion of erythroid progenitors. (a) Number of surviving cells after culturing for 72 hr with EPO and SCF. Dead cells were determined using 7-AAD staining. n = 6 in each group. (b) Representative scatter plot image of the expression of cKit and Lin markers on surviving cells after culture for 72 hr with EPO and SCF. (c) Number of surviving cells retaining the features of LK cells gated on Sca1(-)IL7RaFc?RII/III(-)CD41(-) after a 72-hr culture. n = 6 in each group. (d) Representative endoglin and CD150 expression profile of surviving cultured cells treated with EPO and SCF with (right) or without (left) 50 ng/ml BMP9 gated on Lin(-)cKit(+)Sca1(-)IL7Ra(-)Fc?RII/III(-))CD41(-). (e and f) Comparison of the frequencies of CFU-E and preCFU-E (e) and preMegE and preGM (f) cells gated on the Lin(-)cKit(+)Sca1(-)IL7Ra(-)Fc?RII/III(-))CD41(-) fraction. Results obtained after culturing the cells for 72 hr with EPO and SCF in the absence or presence of BMP9. *p < .05 versus the absence of BMP9 by the Steel test. The representative dot plots and box plots showing the upper quartile, median value and lower quantile of three independent experiments are shown in (a), (c), (e) and (f)

Fig 3: Single-cell RNA-seq analysis of interfollicular keratinocytes, related to Figure 3WT mice were treated daily with either vehicle control (S. epi + vehicle) or antiretroviral (S. epi + antiretroviral) beginning at 1 week before S. epidermidis association for a total of 3 weeks. At day 14 post association, interfollicular keratinocytes were sorted by FACS from the epidermis of ear pinnae and analyzed by single-cell RNA-seq. (A) UMAP projection plots showing the expression profiles of keratinocytes. Colors represent cells clustered together based on similarity of global gene expression. Cells identity was assigned based on the expression level of specific genes: Interfollicular epidermal basal cells: Krt14hiMt2hiMt1hiPostnlow; Interfollicular epidermal differentiated cells subcluster 1 (D1): Krt14midKrt10midMt4hi; Interfollicular epidermal differentiated cells subcluster 2 (D2): Krt14lowKrt10hiSbsnhi;Interfollicular epidermal keratinized cells subcluster 2 (K2): Krt14lowKrt10hiLorhiFlg2hi; Upper hair follicle: Krt79hiKrt17hiDefbhi; and Infundibular basal cells: Krt14midMt2midMt1midPostn+. (B) Schematic illustrating the anatomical localization of distinct populations of keratinocytes. (C) UMAP projection plots depicting expression of the indicated genes that are involved in antigen presentation on MHC class II. Data are representative of one experiment using 5 mice per group. (D) MLV SU expression (shown as integrated MFI) detected by flow cytometry on the surface of Sca-1+CD49f+MHCII+ and Sca-1+CD49f+MHCII– keratinocytes from unassociated or S. epidermidis-associated mice at day 7 post-association. ****P ? 0.0001 as calculated with one-way ANOVA with Holm-Šidák multiple comparison test (D). Data are representative of one (A and C) or three (D) independent experiments using 5 mice per group.

Fig 4: Type I IFN is required for S. epidermidis induced T cells responses in the skin, related to Figure 1(A) Flow cytometry gating strategy used to identify various T cell subsets and MAIT cells. Tc1 and Tc17 cells are defined as live CD45+ Thy1.2+ TCRß+ ?dTCR– CD8b+ IFN-?+ and live CD45+ Thy1.2+ TCRß+ ?dTCR– CD8b+ IL-17A+, respectively. Th1 and Th17 cells are defined as live CD45+ Thy1.2+ TCRß+ ?dTCR– CD4+ Foxp3– IFN-?+and live CD45+ Thy1.2+ TCRß+ ?dTCR– CD4+ Foxp3– IL-17A+, respectively. ?dTCRlow cells are defined as live CD45+ Thy1.2+ TCRß– ?dTCRlow. MAIT cells are defined as live CD45+ Thy1.2+ TCRß+ MR1-tetramer+. (B) Flow cytometry gating strategy use to identify interfollicular keratinocytes (DAPI– CD45– CD31– CD34– CD49f+ Sca-1+) in single cells suspensions isolated from the epidermis of mouse ear pinnae 7 days post daily S. epidermidis association. (C) Principal component analysis of global gene expression of RNA-seq performed on interfollicular keratinocytes sorted from the epidermis at day 7 post S. epidermidis association. Ellipses denote 95% confidence intervals of the mean. Keratinocytes were isolated from ear pinnae. (D) Absolute number of CD8+ T cells, CD4+ T cells, ?dTCRlow and MAIT cells, and frequency of IL-17A+ or IFN-?+ CD8+ or CD4+ T cells, as well as IL-17A+?dTCRlow and MAIT cells in the ear pinnae of unassociated mice or S. epidermidis-associated mice treated with anti-IFNAR1 or isotype control antibodies. Cells were stimulated with PMA and ionomycin. *P ? 0.05, **P ? 0.01, ***P ? 0.001, ****P ? 0.0001 as calculated with one-way ANOVA with Holm-Šidák multiple comparison test. Data are represented as mean ± SEM. Data are representative of two independent experiments using 4-5 mice per group (with the symbols in (C) and (D) each representing an individual mouse).

Fig 5: Fc?RIIB deficiency impairs the differentiation of gMDSCs from HPCs in the tumor-bearing mice. (A) The percentage of proliferating (Ki67+) MDSCs in WT and KO tumor tissues were analyzed with flow cytometry after Ki67 staining, n = 5. (B) Representative staining and frequencies of AnnexinV+7AAD+ cells in MDSCs from WT and KO tumor tissues were assessed by FCM. (C) The gene expression of MDSC-related chemokines within whole tumor tissues from WT and KO mice were detected by qPCR; n = 4. Data are expressed as means ± SD. (D) WT and KO BMs were treated with GM-CSF/IL-6 (20ng/mL) to induce MDSCs differentiation, MDSCs ratio and numbers were analyzed after 72 hrs. (E) Gating strategy for granulocyte/macrophage progenitors (GMP; Lin-Sca-1-C-kit+CD16/32+CD34+), common myeloid progenitors (CMP; Lin-Sca-1-C-kit+CD16/32intCD34+), megakaryocyte/erythrocyte progenitors (MEP; Lin-Sca-1-C-kit-CD16/32-CD34-), and percentages of these HPCs subpopulations rates in BMs from WT and KO tumor bearing mice were detected, n = 5. (F) Representative photograph of femurs dissected from WT and KO tumor-free or MC38 tumor-bearing mice on day 21 after tumor cells implantation. Data are expressed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant difference, by Mann-Whitney test.