Fig 2: The Effect of LAL on T Cell Fate in Target OrgansAllo-HCT was set up and single cells were isolated on day 13 post-HCT and stained for analysis by flow cytometry, as in Figure 3.(A) The zebra plots and summary graphs show the frequencies of donor necrotic (7-AAD+ annexin V+) on gated CD4 or CD8 T cells isolated from recipient guts.(B–I) Histograms and summary graphs show mean of fluorescence intensity of Ki67 (B), DCFDA(C), BODIPY (D), cpt1a (E), PD-1 (F), lag-3 (G), and cyto-ID (H) and PD-L1 (I).The Kruskal-Wallis test was used for statistical analysis. The experiments were repeated 2 times. The data are presented as means ± SDs. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 3: Mincle is required for TH17 mediated EAE progression.a–c Lymph nodes were harvested from MOG immunized mice on Day 9 post immunization, and cells were cultured with increasing concentrations of MOG for 72 h. Cytokine concentrations in the culture media were measured by ELISA, n = 4 biological replicates. d, e Mean clinical score of EAE mice (n = 5) induced by adoptive transfer of MOG-reactive d TH1 or e TH17 cells. f Brain lymphocytes from TH17 recipient mice were harvested at the peak of disease and analyzed by flow cytometry with indicated antibodies by flow cytometry, n = 5 biological replicates. g H&E staining and Luxol fast blue staining of lumbar spinal cords at peak of the disease in recipient mice. Scale bar, 100 µM. Arrows in the upper panel indicate inflammatory cells infiltration, and arrows in the lower panel indicate demyelination area. Representative data are shown for n = 4. h Percentage of CD45+ CD4+ cells of total CNS infiltrated cells from Rag1-/- mice that received MOG-reactive TH17 cells, n = 4 biological replicates. i Cell numbers of CNS-infiltrating CD4+ T cells from (h, j). Percent of Ki67/CD4 double-positive cells from spinal cords from adoptive transfer experiments 9 days after transfer (representative images shown in Supplemental Fig. 3f.). n = 5, biological replicates. **P < 0.01 (Two-sided student’s t test for a–c, f, h–j). ***P < 0.001 (two-way ANOVA for d, e). Data are represented as mean ± SD. Exact P values for asterisks (from left to right): e <0.0001 f 0.0009 0.0033 0.0037 h 0.0025 i 0.0069 j 0.0015.

Fig 4: Histology of pUR4-treated tumors confirms changes in matrix and reveals a decrease in proliferation. (A) Stained tumor sections show a decrease in fibronectin and collagen in pUR4-treated animals and no changes in R1R2-treated animals. Bars represent 100 µm. (B) Western blots from the tumors confirm a decrease in fibronectin and collagen with pUR4 treatment, but collagen I did not decrease with R1R2 therapy. N=4/4/4/4 replicates/treatment. Relevant pairs were evaluated by t-tests. *P<0.05. (C) Proliferation was diminished after treatment with pUR4. Sections were stained for Ki67. Bars represent 100 µm. N=9/9/9/9. Pairs were evaluated by t-tests. **P<0.01. (D) Apoptosis was not affected in vivo by the various treatments. Sections were stained for TUNEL. Examples are shown. Bars represent 100 µm. N=9/9/9/9. Pairs were evaluated by t-tests. (E) Evaluation of blood vessels shows a decrease in area of CD31+ vessels with pUR4 treatment as well as the number of CD31+aSMA+ or CD31+desmin+ double positive vessels (adjusted to the area). Sections from representative tumors with bars representing 100µm. These are intratibial tumors from mice treated for 10 days with the peptides. N=12/12/11/12 for CD31 alone or with a-smooth muscle actin (aSMA) and 11/10/7/7 for staining with desmin. Pairs were compared by t-test. *P<0.05, ****P<0.0001.

Fig 5: In vitro effects of pUR4 and R1R2. (A) Neither pUR4 nor R1R2 affect cell proliferation as evidenced by Ki67 staining after addition of 10 µM of either peptide and evaluating proliferation after 24 hours by flow cytometry. N=5/5/5/5 replicates for each treatment. (B) Similarly, neither pUR4 nor R1R2 increase apoptosis as evidenced by AnnexinV+PI+ by flow cytometry after addition of 10 µM of either. N=4/4/4/4 replicates for each treatment. No differences were detected by t-tests in A or B. (C) pUR4 lowers fibronectin in the matrix of MDA-cancer cells. Matrix (DOC-insoluble fraction) was isolated from cancer cell cultures using MDA-MB-231/B-luc+ for 4 days in the presence of FCS, treated daily with the peptides (10 µM) and evaluated by ELISA. The amount was corrected to total protein measured by the BCA method. N=17/22/12/14 replicates (arranged in the same order as the bars). Comparison by t-test. *P<0.05. (D) Staining of fibronectin (green) and collagen (red) in cultures of 3T3 fibroblasts maintained for 4 days in FCS-containing medium and with daily addition of the peptides shows that pUR4 completely prevented fibronectin matrix assembly. The green seen is intracellular fibronectin. Collagen type I was absent in both pUR4 and R1R2 treated fibroblasts. Bars represent 100 µm. Note that the peptides were added within 1 hour after culturing the cells. (E) The same experiment was performed as in D, but the matrix was obtained (DOC-insoluble fraction) and evaluated by Western blotting for fibronectin and collagen I. The amount of protein was measured by BCA and equivalent protein amounts were applied to the gel, because GAPDH cannot be detected in the DOC-insoluble fraction. Fibronectin diminished only with pUR4 treatment, while collagen I diminished with both treatments. N=15/15/7/7 replicates. (F) Neither pUR4 nor R1R2 modulate mRNA expression of fibronectin and collagen I as measured by qPCR. 3T3 fibroblasts were treated once with 10 µM of the peptides and mRNA isolated 24 hours later. N=12/11/11/11 and 14/11/15/12 replicates (The numbers represent the replicates for each treatment and are arranged in the same order as the bars from left to right).