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: New mutant mouse system identified sOPN, not iOPN, by CD4+ T cells is protective in aGVHD(A) Genomic mutation to generate LSL-sOPN mice. Black and gray boxes indicate Spp1 exons (only the first five exons are shown). The Spp1 signal sequence in exon 2 was replaced by the Il2 signal sequence (57 nt, marked in green text). The targeted mutation allows the generation of sOPN alone without iOPN generation.(B and C) Functional validation of sOPN secretion using BMDMs from WT, LSL-sOPN, and LSL-sOPN;Vav1Cre mice. OPN concentrations in cell culture supernatants (B) and the cytoplasmic fraction (C) were analyzed by ELISA. The NE-PER kit was used to isolate the cytoplasm. Each data point was obtained from a value from one mouse (n = 3).(D) Representative confocal images showing co-localization of OPN (green) with a Golgi body marker, GOLPH4 (red), in LSL-sOPN; Vav1Cre (upper) BMDMs. Nuclei are stained with DAPI (blue). Scale bar, 5 µm.(E–H) Comparison of aGVHD mice that received LSL-sOPN;Vav1Cre (n = 9 mice) or LSL-sOPN (n = 10 mice) CD4+ T cells. Weight loss (E) and survival (F) are shown.(G and H) Comparison of aGVHD mice that received LSL-iOPN;Vav1Cre (n = 4 mice) or LSL-iOPN (n = 4 mice) CD4+ T cells. Weight loss (G) and survival (H) are shown. aGVHD induction was performed as depicted in Figure 1A (E–H). Statistical analyses were performed using unpaired two-tailed t test (B and C) or two-way ANOVA followed by post hoc Sidak multiple comparison tests (E and G). Statistical analysis of survival (F and H) was performed with the Gehan-Breslow-Wilcoxon test. Data are shown as mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001. See also Figure S5.

Fig 4: CD4+ T cell-derived OPN limits T cell infiltration in the gut and cell death in the GI tract during aGVHDaGVHD was induced as described in Figure 1A, and the recipients were analyzed on day 7 after adoptive cell transfer.(A and B) Cell numbers of total CD4+ T (A) and CD44+CD62L-CD4+ T effector (B) cells in SI. n = 4 mice/group.(C–E) Cell numbers of total CD4+ T (C), CD44+CD62L-CD4+ T effector (D), and indicated TH cell subsets (E) in the colon. Results are representative of two independent experiments. One data point denotes a value from one mouse. Recipients of WT (n = 4) or Spp1-/- CD4+ T cells (n = 5).(F–J) Representative images of TUNEL staining in SI (F and G) and colon (H and I), comparing recipients with transferred CD4+ T cells of WT (F and H) versus Spp1-/- (G and I). Scale bar, 50 µm. (J) Quantification of TUNEL+ cell staining in SI and colon. Eight fields per mouse were analyzed, and the average of TUNEL-positive cells per field was denoted as one data point per mouse. SI data are from n = 4 mice with WT CD4+ T cells and n = 3 mice with Spp1-/- CD4+ T cells. Colon data are from n = 3 mice for both groups. Statistical analyses were performed using an unpaired two-tailed t test (A–D) or two-way ANOVA (E and J) followed by a post hoc Sidak multiple comparison test with repeated measures. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

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.