Table1_ICOSLG-associated immunological landscape and diagnostic value in oral squamous cell carcinoma: a prospective cohort study.XLSX

Background: We previously reported that stroma cells regulate constitutive and inductive PD-L1 (B7-H1) expression and immune escape of oral squamous cell carcinoma. ICOSLG (B7-H2), belongs to the B7 protein family, also participates in regulating T cells activation for tissue homeostasis via binding to ICOS and inducing ICOS+ T cell differentiation as well as stimulate B-cell activation, while it appears to be abnormally expressed during carcinogenesis. Clarifying its heterogeneous clinical expression pattern and its immune landscape is a prerequisite for the maximum response rate of ICOSLG-based immunotherapy in a specific population.Methods: This retrospective study included OSCC tissue samples (n = 105) to analyze the spatial distribution of ICOSLG. Preoperative peripheral blood samples (n = 104) and independent tissue samples (n = 10) of OSCC were collected to analyze the changes of immunocytes (T cells, B cells, NK cells and macrophages) according to ICOSLG level in different cellular contents.Results: ICOSLG is ubiquitous in tumor cells (TCs), cancer-associated fibroblasts (CAFs) and tumor infiltrating lymphocytes (TILs). Patients with high ICOSLGTCs or TILs showed high TNM stage and lymph node metastasis, which predicted a decreased overall or metastasis-free survival. This sub-cohort was featured with diminished CD4+ T cells and increased Foxp3+ cells in invasive Frontier in situ, and increased absolute numbers of CD3+CD4+ and CD8+ T cells in peripheral blood. ICOSLG also positively correlated with other immune checkpoint molecules (PD-L1, CSF1R, CTLA4, IDO1, IL10, PD1).Conclusion: Tumor cell-derived ICOSLG could be an efficient marker of OSCC patient stratification for precision immunotherapy.</p

H-MSCs^WT reduced infarct size and collagen I expression 7 days post-MI.

<p>(A) Representative Masson's trichrome staining of heart tissue to show the infarct area (blue) 7 days after MI. (B) Representative immunofluorescence staining of collagen I (red) in LV. (C) Quantification of cardiac infarct size in A. (D) Quantification of collagen I percentage of LV in B. n = 5–6, *P<0.05 vs. others.</p

AT2R Stimulation Enhanced Survival of Transplanted BMMNCs in the Region of Ischemic Myocardium <i>in vivo</i>.

<p>Donor BMMNCs derived from males rats were injected into female rats intramyocardially. 3 days after injection, the abundance of sry gene in ischemic (A) and non-ischemic region (B) of heart tissue was determined by real-time PCR. BMMNCs group n=3; and other groups n=4. *<i>P</i> < 0.05 versus BMMNCs group, and ^#<i>P</i> < 0.05 versus BMMNCs+AngII+Valsartan+PD123319 group.</p

Leptin deficit hypoxia exposed-MSCs failed to limit cardiac fibroblast activation, through TGF-β/Smad2 and MRTF-A.

<p>(A) Western blot of TGF-β, p-Smad2, MRTF-A and MRTF-B in cellular lysates from hypoxia-activated cardiac fibroblasts co-cultured with H-MSCs^WT and H-MSCs^ob/ob. Blots were reprobed for α-Tublin as a control. (B) Densitometric analysis of protein level expressed as % of controls (N-CFs). (C) Immunofluorescence staining of MRTF-A and DAPI has done in H-CFs from four groups. * P<0.05 versus N-CF; #P<0.05 versus H-CF-H-MSCs. Scale bars in E represent 50 µm.</p

H-MSCs^WT prevent hypoxia induced cardiac fibroblast activation, but leptin deficit inhibit this effect.

<p>(A) Western blot of α-SMA, collagen IαI and SM22αin cellular lysates from hypoxia-activated cardiac fibroblasts co-cultured with H-MSCs^WT and H-MSCs^ob/ob. Blots were reprobed for α-Tublin as control. (B) Quantification analysis of α-SMA and SM22α protein level expressed as % of controls (N-CFs). (C) Immunofluorescence staining of α-SMA in hypoxia-activated cardiac fibroblasts co-cultured with H-MSCs^WT and H-MSCs^ob/ob. (D) Quantitative analysis of α-SMA immunofluorescence staining presented on (C). (E) Quantitative analysis of collagen protein level expressed as % of controls (N-CFs) in A. (F) Quantitative analysis of hypoxia-activated cardiac fibroblasts co-cultured with H-MSCs^WT and H-MSCs^ob/ob, stained with Sirius Red. (G) Western blot of α-SMA in cellular lysates of hypoxia-activated wide type or leptin deficit db/db cardiac fibroblasts co-cultured with H-MSCs^WT. (H) Quantification analysis of α-SMA protein level expressed as % of controls (N-CFs). * P<0.05 versus N-CF; #P<0.05 versus H-CFs-H-MSCs. Scale bars in C represent 50 µm.</p

H-MSCs^WT reverses hypoxia stimulated collagen production and cardiac fibroblast differentiation into myofibroblasts.

<p>H-MSCs^WT and N-MSCs^WT co-cultured with H-CFs at normal culture condition 24 hrs. Cardiac fibroblasts from the lower chamber which co-cultured with different preconditioned MSCs^WT were collected for subsequent experiments. (A) Representative western blot of α-smooth muscle actin, collagen IαI and SM22α in cellular lysates from cardiac fibroblasts co-cultured with N-MSCs^WT and H-MSCs^WT. (B) Quantification analysis of western blot in A. (C) Immunofluorescence staining for α-smooth muscle actin in H-CFs co-cultured with N-MSCs^WT and H-MSCs^WT. (D) Quantification analysis of images presented on (C). (E) Quantitative analysis of the effects of N-MSCs^WT and H-MSCs^WT on the collagen production induced by hypoxia in cardiac fibroblasts stained by Sirius Red, resolution in 1 mol HCl. Data are the mean ± SEM of three independent experiments. * P<0.05 versus H-CF; #P<0.05 versus H-CF-N-MSCs^WT. Scale bars in C represent 50 µm.</p

H-MSCs^WT decrease myofibroblasts in peri-infarction region.

<p>(A) Immunofluorescence staining of myofibroblast (DAPI (blue), α-SMA (red) and FSP (green)) in heart frozen section. Arrows indicate double positively stained myofibroblasts. Scale bars represent 50 µm. (B) Quantification of double positive stained myofibroblasts in A, analyzed by Image Pro 6.0 software.*P<0.05 versus N-MSCs, #P<0.05 versus H-MSCs, n≥5 in each group.</p

Transplantation of AT2R Stimulated BMMNCs Improved Global Heart Function and Reduced Infarct Size.

<p>(A) Representative M-mode images showing cardiac function in each group. (B) Ejection Fraction; (C) Fraction Shortening; (D) LVIDd; and (E) LVIDs. Sham n=5; DMEM group n=8; BMMNCs group n=9; BMMNCs + AngII + Valsartan group n=10; BMMNCs +CGP42112A group n=10; and BMMNCs + AngII + Valsartan + PD123319 group n=9. *<i>P</i> <0.05 versus DMEM group and BMMNCs group, #P < 0.05 versus BMMNCs + AngII + Valsartan +PD123319 group. (F) Intramyocardial transplantation of AT2R stimulated BMMNCs reduced infarct size. DMEM group n=4; BMMNCs group n=5; BMMNCs+AngII+Valsartan group n=6; BMMNCs+CGP42112A group n=5; and BMMNCs+AngII+Valsartan+PD123319 group n=4. *<i>P</i> < 0.05 versus DMEM group and BMMNCs group, ^#<i>P</i> < 0.05 versus BMMNCs+AngII+Valsartan+PD123319 group.</p

Effect of AT2R Activation on Cardiomyocyte Protection of BMMNCs <i>in vitro</i>.

<p>(A to F) BMMNCs were initially pre-incubated with DMEM, Ang II, CGP42112A, AngII+Valsartan, or AngII+Valsartan+PD123319 for 2 hours at 37°C, respectively. Then preconditioned BMMNCs were co-cultured with NRCMs under hypoxia in serum free medium for 48 hours. Apoptotic NRCMs were detected using TUNEL assay. Bar=100μm; Green represents TUNEL positive cells; Red represents Troponin T (TnT); Blue represents nuclei; and yellow arrowhead represents apoptotic NRCMs. (G) Quantification of apoptotic NRCMs. n=3 for each group; *<i>P</i> < 0.05 versus NRCMs single culture group and BMMNCs group, ^#<i>P</i> < 0.05 versus BMMNCs+AngII+Valsartan+PD123319 group.</p

Hypoxia Preconditioned Mesenchymal Stem Cells Prevent Cardiac Fibroblast Activation and Collagen Production via Leptin

<div><p>Aims</p><p>Activation of cardiac fibroblasts into myofibroblasts constitutes a key step in cardiac remodeling after myocardial infarction (MI), due to interstitial fibrosis. Mesenchymal stem cells (MSCs) have been shown to improve post-MI remodeling an effect that is enhanced by hypoxia preconditioning (HPC). Leptin has been shown to promote cardiac fibrosis. The expression of leptin is significantly increased in MSCs after HPC but it is unknown whether leptin contributes to MSC therapy or the fibrosis process. The objective of this study was to determine whether leptin secreted from MSCs modulates cardiac fibrosis.</p><p>Methods</p><p>Cardiac fibroblast (CF) activation was induced by hypoxia (0.5% O₂). The effects of MSCs on fibroblast activation were analyzed by co-culturing MSCs with CFs, and detecting the expression of α-SMA, SM22α, and collagen IαI in CFs by western blot, immunofluorescence and Sirius red staining. In vivo MSCs antifibrotic effects on left ventricular remodeling were investigated using an acute MI model involving permanent ligation of the left anterior descending coronary artery.</p><p>Results</p><p>Co-cultured MSCs decreased fibroblast activation and HPC enhanced the effects. Leptin deficit MSCs from Ob/Ob mice did not decrease fibroblast activation. Consistent with this, H-MSCs significantly inhibited cardiac fibrosis after MI and mediated decreased expression of TGF-β/Smad2 and MRTF-A in CFs. These effects were again absent in leptin-deficient MSCs.</p><p>Conclusion</p><p>Our data demonstrate that activation of cardiac fibroblast was inhibited by MSCs in a manner that was leptin-dependent. The mechanism may involve blocking TGF-β/Smad2 and MRTF-A signal pathways.</p></div