Additional file 1 of Neurons upregulate PD-L1 via IFN/STAT1/IRF1 to alleviate damage by CD8+ T cells in cerebral malaria

Supplementary Material 1: Fig. S1. Nerve cell injury and activated CD8+ T cell infiltration in the ECM mouse brain. A) H&E staining of ECM brains showed multiple spots of intracerebral hemorrhage (dark blue arrow). B) IHC staining of synaptophysin (light blue arrow) in the cerebrum of control and ECM mice. C) Nissl staining of neurons (pink arrow) in the brainstem of control and ECM mice. Data are expressed as mean ± SD; n = 8 fields per group. D) IF staining of TUNEL+ cells in the olfactory bulb, cerebrum, cerebellum, and brainstem of control and ECM mice. E) IF staining of LC3 in neurons in the cerebrum of control and ECM mice. F) IF staining of Ki67+CD8+ T cells (yellow arrow) in the olfactory bulb of ECM mice. Fig. S2 The interaction of neurons and ECM CD8+ T cells in vitro. A) IF staining of naïve or ECM CD8+ T cells (yellow arrow) adhering to neurons (left image) and quantification of adhered CD8+ T cells (right image). Data are expressed as mean ± SD; unpaired t-test, n > 3 sections per group. B) CCK-8 detection in the supernatant of neurons treated with different proportions of CD8+ T cell culture supernatant. Data are expressed as mean ± SD; unpaired t-test, n = 4 per group. C) Flow cytometry of JC-1 (FL-1: monomer, FL-2: J-aggregates) in neurons co-cultured with ECM CD8+ T cell. D) IF staining of ECM CD8+ T cell (yellow arrow) adhering to axon. E) q-PCR detection of the H2-D1 expression in neurons co-cultured with ECM CD8+ T cell. Data are expressed as mean ± SD; unpaired t-test, n = 3 per group. F) Flow cytometry of the H2-D/K levels on neurons co-cultured with ECM CD8+ T cell. G) IF staining of H2-D/K and CD18 in CD8+ T cell (yellow arrow) and neuron (white arrow) co-culture system. Fig. S3 IFNβ or IFNγ induces neurons to upregulate PD-L1. A) IHC staining of PD-L1 in the olfactory bulb, cerebrum, and cerebellum of control and ECM mice (red arrow: PD-L1+ nerve cells). B) q-PCR analysis of Cd274 expression in neurons with IFNβ (100 U/mL, the same below) or IFNγ (20 ng/mL, the same below) stimulation at different points in time. Data are expressed as mean ± SD; unpaired t-test, n = 3 per group. C) WB analysis of PD-L1 levels in neurons with IFNβ stimulation at different points in time. D) IF staining of PD-L1 in neurons stimulated with IFNβ or IFNγ. Fig. S4 IFNβ or IFNγ induces the expression of PD-L1 through the STAT1/IRF1 pathway in neurons. RNA-seq was performed on neurons stimulated with IFNγ (20 ng/mL, the same below) or ECM CD8+ T cells for 24 h. A) The Gene Ontology enrichment dot plot of IFN-related pathways in neurons stimulated with ECM CD8+ T cells. B) Violin plots of Cd274, Stat1, and Irf1 expression in neurons with IFNγ or ECM CD8+ T cell stimulation (Padj < 0.05). C) q-PCR detection of Stat1 and Irf1 expression in neurons stimulated at different times with IFNβ (100 U/mL, the same below) or IFNγ. Data are expressed as mean ± SD; unpaired t-test, n = 3 per group, ****P < 0.0001. D) WB analysis of p-STAT1 in neurons stimulated at different times with IFNβ or IFNγ. E) IF staining of p-STAT1 in neurons with IFNβ or IFNγ stimulation. F) q-PCR detection of Cd274, Stat1, and Irf1 in neurons treated with fludarabine (20 µM), under IFNβ or IFNγ stimulation. Data are expressed as mean ± SD; unpaired t-test, n = 3 per group. G) Flow cytometry of PD-L1 on neurons treated with fludarabine, IFNβ or IFNγ. H) WB analysis of p-STAT1, IRF1, and PD-L1 in neurons treated with IFNγ and different doses of fludarabine. I) IHC staining of IRF1 in the olfactory bulb, cerebrum, and cerebellum of control and ECM mice. Fig. S5 Changes of the Ifnar1−/− or Ifngr1−/− neurons in ECM brain or in vitro treated with IFNs. A) Flow cytometry of PD-L1 on Ifnar1+/− and Ifnar1−/− neurons treated with IFNβ or IFNγ. B) Flow cytometry of PD-L1 on Ifngr1+/− and Ifngr1−/− neurons treated with IFNγ or ECM CD8+ T cells. C) Statistical results of WB analysis of p-STAT1, IRF1, and PD-L1 levels in Ifnar1+/+ or Ifnar1−/−, and Ifngr1+/+ or Ifngr1−/− neurons treated with IFNβ or IFNγ. Data are mean ± SD; unpaired t-test, n ≥ 3 per group. D) Statistical results of WB analysis of p-STAT1, IRF1, and PD-L1 levels in the brainstem of Ifnar1+/+ or Ifnar1−/−, and Ifngr1+/− or Ifngr1−/− mice with PbA infection, uninfected littermates as control. Data are expressed as mean ± SD; unpaired t-test, n ≥ 3 per group. E) IF staining of p-STAT1, IRF1, and PD-L1 in the brainstem of Ifnar1+/+ and Ifnar1−/− mice infected with PbA (yellow arrow: positive cell). F) IF staining of p-STAT1, IRF1, and PD-L1 in the brainstem of Ifngr1+/+ and Ifngr1−/− mice infected with PbA (yellow arrow: positive cell). G-I) IF staining of PD-L1, p-STAT1, and IRF1 in the brainstem of Ifngr1+/+ and Ifngr1−/− mice infected with PbA, neurons tagged with NeuN (yellow arrow: neuron). J) IF staining of the infiltrating CD8+ T cells (yellow arrow) in the brains of Ifngr1−/− mice infected with PbA. K) IF staining of the infiltrating IFNγ+CD8+ T cells (yellow arrow) in the brainstem of Ifngr1−/− mice infected with PbA. Fig. S6 Upregulation of PDL1 protects neurons in vitro and in ECM brain. A) Optical microscope images of the Ad: PD-L1 or Ad: IgGFc treated neurons stimulated with ECM CD8+ T cells. B) IF staining of NeuN in Ad: PD-L1 or Ad: IgGFc treated neurons stimulated with ECM CD8+ T cells. C) Flow cytometry of PI+ neurons in Ad: PD-L1 or Ad: IgGFc treated neurons stimulated with ECM CD8+ T cells. D) The diagram of stereotaxic microinjection of the brainstem in mice (red dot: injection location). E) H&E staining on the Ad: PD-L1 injected side or non-injected side of ECM brainstem (yellow arrow: hemorrhage). F) IF staining of PD-L1 on the Ad: PD-L1 injected side or non-injected side of ECM brainstem.