adult human dermal lecs  (PromoCell)

Journal: The Journal of Clinical Investigation

Article Title: Organ-specific features of human kidney lymphatics are disrupted in chronic transplant rejection

doi: 10.1172/JCI168962

Figure Lengend Snippet: ( A ) Violin plots showing upregulation of IFN-inducible genes IFITM2 and IFITM3 in LECs from rejecting allografts. ( B ) UMAP of the scRNA-Seq data showing enrichment of IFN-γ within the T/NK cell cluster. ( C ) UMAP showing enrichment of an IFN-γ signature, including IFNGR1 , IFNGR2 , IFITM2 , and IFITM3 . ( D ) CellPhoneDB interaction map depicting predicted lymphatic-CD4 + T cell crosstalk in rejection. Inhibitory interactions (blue) include PVR and LGALS9; stimulatory interactions (red) are also shown. Node size reflects expression frequency; line intensity indicates interaction strength. Ligands of interest, PVR and LGALS9 , are highlighted. ( E ) Heatmap of immune checkpoint interactions between LECs and effector CD4 + T cells across disease states. Color indicates normalized CellPhoneDB interaction score. All scores were normalized for each ligand-receptor pair. ( F ) Immunofluorescence validation of PVR expression on PDPN + lymphatics (arrowhead) in rejecting kidneys ( n = 2); CD4 + T cell shown in contact (asterisk). Scale bar: 30 μm. ( G ) IFN-γ stimulation of cultured human LECs increases LGALS9 levels at 24 and 48 hours (qPCR; *** P = 0.0002, ** P = 0.0093, respectively) relative to HPRT. ( H ) LGALS9 protein secretion increased at 48 and 72 hours (ELISA; *** P = 0.0002, **** P < 0.0001, respectively) after IFN-γ stimulation of cultured human LECs. qPCR and ELISA experiments were repeated 3 times, and all assays were performed in duplicate, with each dot on the graph representing the mean data obtained for each repeat.

Article Snippet: Adult human dermal LECs (PromoCell, C-12217) were cultured in MV2 medium and treated with recombinant human IFN-γ (50 ng/mL) or unstimulated control medium for 24, 48, or 72 hours.

Techniques: Expressing, Immunofluorescence, Biomarker Discovery, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Associated Lymphatics Upregulate MHC-II to Suppress Tumor-Infiltrating Lymphocytes

doi: 10.3390/ijms232113470

Figure Lengend Snippet: Peritumoral LECs upregulate MHC-II, PD-L1, and various co-inhibitory molecules compared to naïve dermal LECs. ( A ) Schematic of experimental design analyzing naïve dermal LECs in the ear and peritumoral LECs of the same mice. ( B ) Quantification of MHC-II and PD-L1 positivity in naïve and peritumoral LECs (CD45- podoplanin+ CD31+) at day 7, 14, and 21 post B16F10 tumor inoculation in WT (C57/BL6) mice. ( C ) Quantification of MHC-II and PD-L1 positivity in naïve and peritumoral LECs at day 14 post E0771 tumor inoculation in WT mice. ( D ) Quantification of MHC-II and PD-L1 positivity in naïve and peritumoral LECs in MMTV-PyMT mice with spontaneous mammary tumors. ( E ) Quantification of PD-L2, HVEM, and CD48 positivity in naïve and peritumoral LECs at day 21 post B16F10 tumor inoculation in WT mice. ( F ) Representative immunofluorescence image stained for LYVE-1 (white) and MHC-II (green) in peritumoral and skin margin lymphatics in WT mice at day 14 post B16F10 inoculation. DAPI nuclear staining in blue. Scale bars, 100 μm ( left ), 20μm ( top right and bottom right ). Statistical significance assessed using paired student’s t test on data from at least 3 biological replicates; error represented as SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Human dermal LECs (PromoCell, Cat#C-12217; Heidelberg, Germany) were grown to be 60–80% confluent in endothelial growth medium-MV-2 (PromoCell, Cat#C-22022) before treatment with cytokines and growth factors.

Techniques: Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Associated Lymphatics Upregulate MHC-II to Suppress Tumor-Infiltrating Lymphocytes

doi: 10.3390/ijms232113470

Figure Lengend Snippet: IFNγ induces upregulation of MHC−II in LECs in vitro and in vivo in the tumor periphery. ( A – D ) Relative mRNA expression of HLA−DRA, HLA−DM, PD−L1, LYVE−1 in cultured human dermal LECs after 24-h treatment with ( A ) IFNγ (100 ng/mL), ( B ) VEGF−C (100 ng/mL), ( C ) IL−2 (10 ng/mL) and ( D ) TNF−α (10 ng/mL). ( E ) Representative FACS histogram plot and ( F ) quantification of MHC−II and PD−L1 positive human dermal LECs in culture after 24-hour treatment with IFNγ (100 ng/mL) as measured by flow cytometry. ( G ) Representative FACS histogram plots and ( H ) quantification of MHC-II and PD-L1 positive peritumoral LECs in WT mice treated with IFNγ neutralizing antibody or IgG isotype control at day 14 post tumor inoculation. Statistical significance assessed using unpaired student’s t test on data from at least 3 biological replicates for cell culture studies and at least 4 biological replicates for mouse experiments; statistical analysis not performed on ( B – D ) for which at least 2 biological replicates were used; error represented as SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001. n.d, not detected mRNA in treated group.

Article Snippet: Human dermal LECs (PromoCell, Cat#C-12217; Heidelberg, Germany) were grown to be 60–80% confluent in endothelial growth medium-MV-2 (PromoCell, Cat#C-22022) before treatment with cytokines and growth factors.

Techniques: In Vitro, In Vivo, Expressing, Cell Culture, Flow Cytometry, Control

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Associated Lymphatics Upregulate MHC-II to Suppress Tumor-Infiltrating Lymphocytes

doi: 10.3390/ijms232113470

Figure Lengend Snippet: Cultured LECs engulf tumor debris and induce CD8+ T cell proliferation with reduced effector function. ( A ) Representative confocal micrograph including Z-plane of CFSE-labeled B16F10 tumor debris (arrows) within cultured mouse dermal LECs (MDLEC) and human dermal LECs (HDLEC). Scale bars; left, 10 μm; right, 7 μm. ( B ) CD4+ OT-II and ( C ) CD8+ OT-I proliferation after co/tri-culture conditions with the indicated cell types in the presence of LEC or DC unpulsed or pulsed with OVA protein (2 mg/mL) 24 h prior to co/tri-culture. ( D ) Representative FACS plot and ( E ) quantification of GZMb+ proliferated OT-I cells after culture with indicated cell types in the presence of LEC or DC either unpulsed or pulsed with OVA protein (2 mg/mL) 24 h prior to co/tri-culture. ( F ) OT-I proliferation after co/tri-culture with indicated cell types either NH 4 -Cl pretreated or not for 24 h followed by pulsing or no pulsing with OVA protein (2 mg/mL) 24 h prior to co/tri-culture. ( G ) OT-I proliferation after culture with indicated cells pulsed or not pulsed with OVA protein (2 mg/mL) at 4 °C 24 h prior co/tri-culture. Statistical significance assessed using one-way ANOVA on data from at least 3 biological replicates (B-G); DC-OVA, OVA pulsed DC; LEC-OVA, OVA pulsed LEC; NLEC, NH 4 -Cl pretreated LEC; NLEC-OVA, OVA pulsed NH 4 -Cl pretreated LEC..Error represented as SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: Human dermal LECs (PromoCell, Cat#C-12217; Heidelberg, Germany) were grown to be 60–80% confluent in endothelial growth medium-MV-2 (PromoCell, Cat#C-22022) before treatment with cytokines and growth factors.

Techniques: Cell Culture, Labeling