Fig 2: CD4+ T cell-derived OPN is protective in aGVHD(A) Schematic diagram of aGVHD induction. Recipient mice were BALB/c Spp1−/−, lethally irradiated before transferring B6 CD4+ T cells of various genotypes and T cell-depleted Spp1−/− B6 BM cells. This protocol was used for almost all the experiments unless otherwise noted.(B–G) Comparison of aGVHD severity between recipients with WT (n = 13) or Spp1−/− (n = 14) CD4+ T cells. Weight loss (B), clinical score (C), and survival (D) were evaluated. Data are combined from three independent experiments. Representative images of hematoxylin-eosin (H&E) staining of the colon (E) and SI (F) with histology scores (G) on day 7 after adoptive cell transfer.Recipients of WT or Spp1−/− CD4+ T cells (n = 4 mice/group), alongside control mice (irradiation only) (n = 2 mice/group), were compared. Insets in (E) depict (1) crypt regeneration, (2) crypt loss, and (3) lamina propria infiltrates in the colon from Spp1−/− CD4+ T cell recipients. Scale bars, 100 μm (10 μm for insets 1–3). Quantitative data were analyzed using two-way ANOVA followed by post hoc Sidak multiple comparison test (B and C) or Tukey’s multiple comparison test (G) with repeated measures. Percentage survival (D) was analyzed by the Gehan-Breslow-Wilcoxon test. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: CD4+ T cell-mediated OPN correlates with increased commensal bacteria, Akkermansia, and reduced levels of epithelium-associated Bacteroidetes(A–G) Bacterial 16S rRNA sequencing was performed to identify bacterial microbiome. aGVHD was induced as depicted in Figure 1A. Fecal samples were obtained 7 days after adoptive cell transfer. Control groups include naive BALB/c WT and Spp1−/− mice, in addition to irradiated BALB/c Spp1−/− mice without any adoptive cell transfer. Data presented are principal coordinate analysis (PCoA) plots (A), the Shannon diversity index (B), and the relative abundance of bacteria (C–G). Frequencies indicated are at bacterial order (C and F) and genus (D, E, and G) levels. Naive WT and irradiated mouse groups, n = 3; naive Spp1−/− mouse group, n = 10; GVHD with WT CD4+ T cells, n = 3; GVHD with Spp1−/− CD4+ T cells, n = 4. Statistical analyses (A–G) were performed as described in the STAR Methods. Adjusted p values from differential abundance analysis; **padj < 0.002.(H) Host gene expression in the SI from mice with aGVHD, induced as shown in Figure 1A, at 7 days after adoptive T cell transfer. cDNA from five mice were pooled as one sample, and error bars (too short to see) were calculated as RQ-MIN and RQ-MAX, as described in STAR Methods. Two independent experiments showed similar results.(I) Bacterial burdens, on day 7 after adoptive T cell transfer, determined by 16S rRNA gene qPCR at the phylum level in SI tissue samples from mice with aGVHD, induced as shown in Figure 1A. Data are shown as mean ± SEM and statistical analyses were performed with two-way ANOVA. One data point denotes a value from one mouse (A, B, D–G, and I). *p < 0.05, ****p < 0.0001. See also Tables S1 and S2.

Fig 4: Overview of the crosstalk networks between developing NC/NP and vertebral chondrocytes during early IVD formation. A) Overview of the cellular network between developing NC/NP cells and vertebral chondrocytes during human early IVD formation. Dots indicate cell subpopulations. The dot size indicates the relative quantity of each subpopulation. The thickness of the directed line indicates the relative quantity of significant ligand–receptor pairs between any two pairs of subpopulations. B) Dot plot showing the communication probability of the indicated ligand–receptor pairs between four vertebral chondrocyte subclusters (sending signals) and seven NC/NP subclusters (accepting signals). C) Dot plot showing the communication probability of the indicated ligand–receptor pairs between seven NC/NP subclusters (sending signals) and four vertebral chondrocyte subclusters (accepting signals). D) UMAP plots showing the expression of SPP1 and CD44 on the UMAP. E) Representative IF images of SPP1 in human axial skeleton sections at the indicated developmental stages. Orange dotted squares are magnified under each image. The white dotted line indicates the presumptive OAF. The asterisks indicate the presumptive IAF. AF, annulus fibrosus; OAF, outer annulus fibrosus; IAF, inner annulus fibrosus; VB, vertebral body; NC, notochord; NP, nucleus pulposus; OC, ossification center. Scale bar, 100 µm. F) Representative IF images of CD44 in human axial skeleton sections at the indicated developmental stages. Scale bar, 500 µm. G) Representative IF images of EMCN in human axial skeleton sections at the indicated developmental stages. The arrows indicate the immunofluorescent signals surrounding the VB. The asterisks indicate the cartilage canals. AF, annulus fibrosus; VB, vertebral body; NP, nucleus pulposus; OC, ossification center. Scale bar, 500 µm. H) Western blot results showing the expression of Vegfa, Cd9, Hif1a and Sox9 in ATDC5 chondrogenic progenitor cells after rSpp1 treatment. I) Representative IHC images of Spp1 in control and degenerated rat IVDs. Scale bar, 200 µm.

Fig 5: Donor CD4+ T cell-derived OPN is protective even with host-derived OPN(A–C) Comparison of aGVHD severity between WT recipients with WT (n = 4) or Spp1−/− (n = 4) CD4+ T cells. Experimental schematics (A), weight loss (B), and survival (C) are shown. Results are representative of three independent experiments.(D–F) Comparison of aGVHD severity between WT and Spp1−/− recipients. Experimental schematics depicting Spp1−/− CD4+ T cells and T cell-depleted Spp1−/− BM being transferred to indicated recipients (D). Weight loss (E) and survival (F) are shown.Results are combined from two independent experiments using n = 12 mice/group for both groups. Statistical analyses were performed using two-way ANOVA followed by a post hoc Sidak multiple comparison test (B, C, and E) with repeated measures. Percentage survival (F) was analyzed by the Gehan-Breslow-Wilcoxon test. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S3.