Journal: bioRxiv

Article Title: HLA-E and NKG2A Mediate Resistance to M. bovis BCG Immunotherapy in Non-Muscle-Invasive Bladder Cancer

doi: 10.1101/2024.09.02.610816

Figure Lengend Snippet: A , Representative multiplexed immunofluorescence (IF) analysis by PhenoCycler TM (also known as CODEX) of bladder tumor sections from one patient with BCG-unresponsive NMIBC. Representative staining of the entire section is shown for presence of stroma (vimentin, blue) and tumor (S100A4, yellow). Scale bar indicates 2mm. B, Magnified inset from Panel A, highlighting tumor HLA-E expression of and use of digital pathology software to identify NKp46 + NK and CD8 T cells with and without co-expression of NKG2A +/-PD-1. Left image scale bar indicates 400μm and right image scale bar indicates 50μm. C, Magnified inset from Panel B, right image, highlighting individual cells expressing CD8, PD-1, and NKG2A. Scale bars indicate 20μm. D, Representative multiplexed immunohistochemistry (IHC) of bladder tumor from one patient with BCG-unresponsive NMIBC measuring expression of pan-cytokeratin (pan-CK), HLA-E, CD3, and NKG2A. Scale bar indicates 100μm. E, Representative digital pathology analyses identifying tumor (red) and adjacent/non-tumor (green) tissue along with exposed areas of glass (yellow) to be excluded from subsequent analyses. Scale bar indicates 1mm. F, Density map highlighting regions of high HLA-E tumor expression. Blue-red color scale indicates HLA-E expression intensity. Scale bar indicates 1mm. G, Summary analysis of frequency of tumor cells that are HLA-E-bright (green) and HLA-E-dim/negative (dim/neg, yellow) in BCG-naïve (N=17) and BCG-unresponsive (unresp., N=24) NMIBC tumors. H, Representative digital pathology analysis on one BCG-naïve (top row) and one BCG-unresponsive (bottom row) NMIBC tumor section highlighting nuclear expression of DAPI (blue) and identification of CD3 + NKG2A + CD8 T cells (black), CD3-NKG2A + NK cells (red), and tumor cells with bright (green) or dim/negative (yellow) expression of HLA-E. I, Proximity analysis (N=41) measuring cell distance (0-150μm) from HLA-E-bright and HLA-E-dim/negative tumors by NKG2A+ NK cells (left side) and NKG2A+ T cells (right side). ****, p<0.00001. P-values were assessed via independent two-sided t-test. J, Representative proximity ligation assay (PLA) using immunofluorescence to profile interactions between HLA-E and NKG2A in one BCG-naïve (top row) and one BCG-unresponsive (bottom row) patient with NMIBC. K, Summary analysis of PLA measuring interactions between HLA-E and NKG2A. P-values were assessed via independent two-sided t-test.

Article Snippet: Briefly, after deparaffinization, rehydration, and antigen retrieval, slides were blocked with Block NT blocking solution (Navinci, NT.1.100.01) for 60 min at 37 °C in a preheated humidity chamber and then incubated with mouse anti-HLA-E (clone: MEM-E/02, Abcam, 1:200) and rabbit anti-NKG2A (clone: EPR23737-127, Abcam, 1:2000) diluted in Diluent 1 NT solution (Navinci, NB.1.100.02) overnight at 4°C.

Techniques: Immunofluorescence, Staining, Expressing, Software, Immunohistochemistry, Proximity Ligation Assay

Journal: bioRxiv

Article Title: HLA-E and NKG2A Mediate Resistance to M. bovis BCG Immunotherapy in Non-Muscle-Invasive Bladder Cancer

doi: 10.1101/2024.09.02.610816

Figure Lengend Snippet: A , UMAP visualization of cell lineages from single-cell RNA sequencing analysis of urothelial tumor samples (N=9, 65,324 cells). B, Heatmap summary of genes most differentially expressed on each cell lineage identified. C, Differentially expressed gene (DEG) analysis of bladder tumor-derived NK cells (left), CD8 T cells (center), and proliferating T cell cycle cells (right) when stratified by high versus dim/negative KLRC1 expression. D and E, UMAP visualizations of bladder tumor-derived NK cells from unsupervised clustering revealing ( D ) five clusters or ( E ) expression of NCAM1 /CD56 for annotation of CD56 BRIGHT and CD56 DIM NK cells (N=2,580 cells). F, Pathway analysis of Hallmark gene networks that are significantly differentially expressed (at least p<0.05) on bladder tumor-derived CD56 BRIGHT and CD56 DIM NK cells. P-values were determined using a two-sided t-test. G and H, UMAP visualization of bladder tumor-derived NK cells ( G ) clustered according to Groups 1-6 defined by Netskar et al. and ( H ) highlighting expression and distribution of representative tissue residency genes. I, Heatmap summary showing average expression for genes associated with tissue residency, chemokines, cytokines, and their receptors on bladder tumor-derived Group 1-6 NK cells (top heatmap) and CD8 T cells and T cell cycle cells when stratified according to KLRC1 expression (bottom heatmap).

Article Snippet: Briefly, after deparaffinization, rehydration, and antigen retrieval, slides were blocked with Block NT blocking solution (Navinci, NT.1.100.01) for 60 min at 37 °C in a preheated humidity chamber and then incubated with mouse anti-HLA-E (clone: MEM-E/02, Abcam, 1:200) and rabbit anti-NKG2A (clone: EPR23737-127, Abcam, 1:2000) diluted in Diluent 1 NT solution (Navinci, NB.1.100.02) overnight at 4°C.

Techniques: RNA Sequencing Assay, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: HLA-E and NKG2A Mediate Resistance to M. bovis BCG Immunotherapy in Non-Muscle-Invasive Bladder Cancer

doi: 10.1101/2024.09.02.610816

Figure Lengend Snippet: A, UMAP visualization of bladder tumor cells from unsupervised clustering revealing seven clusters (B1-B7). B, UMAP visualization of HLA-E expression defined by tertiles, highlighting the top tertile (red dots) as HLA-E-bright (N=5,461) and bottom tertile (blue dots) as HLA-E-dim/negative (N=9,256) tumor cells. C, Pathway analysis of Hallmark gene networks that are significantly differentially expressed (at least p<0.05) on HLA-E-bright and HLA-E-dim/negative bladder tumor cells. P-values were determined using a two-sided t-test. D, Bubble plot showing targeted gene expression across bladder tumor clusters B1-B7 and stratified by HLA-E HIGH or HLA-E LOW tumor clusters. Size of bubble indicates percent of cluster or group and color indicates average gene expression. E, Scatterplot highlighting pseudobulking of bladder tumor cells and expression of HLA-E and ACKR3 (CXCR7). P-value was determined using a two-sided correlation. F, Stacked bar plot showing the frequency of bladder tumor clusters, B1-B7 that are identified in BCG-naïve (green) and BCG-unresponsive (grey) NMIBC tumors. G, Heatmap summary of average gene expression of select chemokines, chemokine receptors, amphiregulin ( AREG ) and EGFR across all major clusters identified by scRNAseq of bladder tumors (N=9, 65,324 cells). H, Ligand-receptor interaction analysis of KLRC1 HIGH cells (NK cells, CD8 T cells, T cell cycle) and HLA-E -bright tumor cells (schematic diagram at top) showing the top 12 ranked ligands by KLRC1-expressing cells and cognate receptors expressed by HLA-E HIGH tumor cells. Ligand-receptor pairs were selected by rank-ordering and using cut-off weight of 0.2. I, Circos plot showing top ranked ligands by KLRC1-expressing cells at the bottom and significant genes expressed by HLA-E HIGH tumor cells as a result of ligand-receptor interactions highlighting putative effects of the HLA-E/NKG2A axis (top) or the IFNG -mediated (middle) or AREG -mediated (bottom) resistance signatures.

Article Snippet: Briefly, after deparaffinization, rehydration, and antigen retrieval, slides were blocked with Block NT blocking solution (Navinci, NT.1.100.01) for 60 min at 37 °C in a preheated humidity chamber and then incubated with mouse anti-HLA-E (clone: MEM-E/02, Abcam, 1:200) and rabbit anti-NKG2A (clone: EPR23737-127, Abcam, 1:2000) diluted in Diluent 1 NT solution (Navinci, NB.1.100.02) overnight at 4°C.

Techniques: Expressing, Targeted Gene Expression

Journal: bioRxiv

Article Title: HLA-E and NKG2A Mediate Resistance to M. bovis BCG Immunotherapy in Non-Muscle-Invasive Bladder Cancer

doi: 10.1101/2024.09.02.610816

Figure Lengend Snippet: A-C , Representative ST-seq images from one BCG-unresponsive NMIBC tumor highlighting ( A ) proximity of NK cells, CD8 T cells, and Tregs to tumor and stromal cells according to HLA-E expression, ( B ) overlaid topographical map of HLA-E tumor/stromal cell expression with color indicating abundance of NK and cytolytic CD8 T cells, and ( C ) proximity of NK cells, CD8 T cells, and Tregs to myeloid cells expressing any combination of CXCL9 , CXCL10 , and/or CXCL11 . D, Correlation analysis of Visium spots, defined as tumor cell with or without stromal cells, for co-expression of CXCL9 and HLA-E expression in BCG-naïve (N=4, left) and BCG-unresponsive (N=4, right) NMIBC tumor specimens. E, Correlation analysis of Visium spots, defined as tumor cell with or without stromal cells, for co-expression of CXCL12 and HLA-E expression in BCG-naïve (N=4, left) and BCG-unresponsive (N=4, right) NMIBC tumor specimens. F, Meta-analysis of all tumor/stroma-labelled Visium spots showing row-scaled expression of pertinent genes stratified by number of neighboring infiltrating NK cells and/or CD8 T-cells. G, Z-scored heatmap showing HLA-E LOW , low-cytotoxic infiltrating tumor spots (N = 1,200 Visium spots) vs HLA-E HIGH and high-NK/CD8 T cell-infiltrated tumor spots (N = 242 Visium spots). H, Representative multiplexed IF analysis by PhenoCycler TM of bladder tumor sections from one patient with BCG-unresponsive NMIBC highlighting the presence of stroma (vimentin, blue) and tumor (S100A4, yellow). Scale bar indicates 2mm. I, Magnified inset from Panel H, highlighting tumor S100A4 expression of and use of digital pathology software to identify NKG2A + NK and CD8 T cells along FoxP3+ CD4+ Tregs, macrophages (CD68 and CD163) and dendritic cells (CD11c and/or HLA-DR). Image scale bar indicates 20μm.

Article Snippet: Briefly, after deparaffinization, rehydration, and antigen retrieval, slides were blocked with Block NT blocking solution (Navinci, NT.1.100.01) for 60 min at 37 °C in a preheated humidity chamber and then incubated with mouse anti-HLA-E (clone: MEM-E/02, Abcam, 1:200) and rabbit anti-NKG2A (clone: EPR23737-127, Abcam, 1:2000) diluted in Diluent 1 NT solution (Navinci, NB.1.100.02) overnight at 4°C.

Techniques: Expressing, Software

Journal: bioRxiv

Article Title: HLA-E and NKG2A Mediate Resistance to M. bovis BCG Immunotherapy in Non-Muscle-Invasive Bladder Cancer

doi: 10.1101/2024.09.02.610816

Figure Lengend Snippet: A, Median fluorescence intensity (MFI) of HLA-E and PD-L1 expression by wild-type (WT) or HLA-E + K562 tumors cultured overnight in the presence or absence of recombinant human IFN-ψ. B and C, Phenograph clustering meta-analysis of tumor-derived and expanded CD56 + CD8 T cells by CyTOF before co-culture with K562 tumors showing B, tSNE analysis of identified CD8 T cell clusters, and C, expression of individual inhibitory and activating receptors by CD8 T cells and their distribution across clusters. D, Representative CyTOF analysis of CD56 and NKG2A expression on tumor-derived NK (top row) and CD8 T cells (bottom row) profiled before (left column) and after (right column) expansion with low dose recombinant human IL-2, IL-7, IL-15 and CD3/CD28 tetramers. E, Representative fluorescence flow cytometric analysis of IFN-g and CD107a expression by CD56 + NK (top row) and CD8 T cells (bottom row) after 6-hour culture alone or in presence of K562 tumor lines with or without pre-treatment with anti-PD-L1 antibodies or with anti-PD-L1 + anti-NKG2A antibodies. F, Fold-change differences between the frequencies of NK and CD56 + CD8 T cells when comparing between experimental conditions (defined by “+”). Individual comparisons are indicated at the bottom. *, p< 0.05; **, p< 0.001. P values determined by paired Wilcoxon matched rank test.

Article Snippet: Briefly, after deparaffinization, rehydration, and antigen retrieval, slides were blocked with Block NT blocking solution (Navinci, NT.1.100.01) for 60 min at 37 °C in a preheated humidity chamber and then incubated with mouse anti-HLA-E (clone: MEM-E/02, Abcam, 1:200) and rabbit anti-NKG2A (clone: EPR23737-127, Abcam, 1:2000) diluted in Diluent 1 NT solution (Navinci, NB.1.100.02) overnight at 4°C.

Techniques: Fluorescence, Expressing, Cell Culture, Recombinant, Derivative Assay, Co-Culture Assay

Journal: BMC Cancer

Article Title: Regulation of KLRC and Ceacam gene expression by miR-141 supports cell proliferation and metastasis in cervical cancer cells

doi: 10.1186/s12885-024-12794-6

Figure Lengend Snippet: Oligonucleotides sequences used for quantifying miR-141 and mRNA of indicated genes

Article Snippet: For staining KLRC1 and KLRC3, the primary antibodies; rabbit polyclonal anti-KLRC1 (Sigma-Aldrich, USA) and mouse monoclonal anti-KLRC3 (Sigma-Aldrich, USA) were used.

Techniques:

Journal: BMC Cancer

Article Title: Regulation of KLRC and Ceacam gene expression by miR-141 supports cell proliferation and metastasis in cervical cancer cells

doi: 10.1186/s12885-024-12794-6

Figure Lengend Snippet: Microarray analysis of transfected HeLa cells with miR-141 overexpressing vector. A Microarray analysis of gene expression in HeLa cells transfected with miR-141 overexpression vs. cells transfected with the control vector using a miRNA library from Exiqon. B Microarray analysis of gene expression in HeLa cells transfected with miR-141 overexpression vs. cells transfected with the control vector using oligonucleotides from Ambion. The blue color indicates the sustained gene expression, the red indicates the upregulated genes, and the green indicates the downregulated genes. C The expression of the most relevant genes to cervical cancer indicates the upregulated genes in red columns and downregulated genes in green. Error bars indicate the STD between Exiqon and Ambion data. D The potential binding sites of miR-141 within the 3 − UTR of KLRC1 and coding sequences of KLRC3, CAM3, and that carried out in-silico by miRWalk online tool

Article Snippet: For staining KLRC1 and KLRC3, the primary antibodies; rabbit polyclonal anti-KLRC1 (Sigma-Aldrich, USA) and mouse monoclonal anti-KLRC3 (Sigma-Aldrich, USA) were used.

Techniques: Microarray, Transfection, Plasmid Preparation, Expressing, Over Expression, Control, Binding Assay, In Silico

Journal: BMC Cancer

Article Title: Regulation of KLRC and Ceacam gene expression by miR-141 supports cell proliferation and metastasis in cervical cancer cells

doi: 10.1186/s12885-024-12794-6

Figure Lengend Snippet: The prediction information between miR-141 and targeted genes identified by microarray analysis using in-silico miRWalk tool

Article Snippet: For staining KLRC1 and KLRC3, the primary antibodies; rabbit polyclonal anti-KLRC1 (Sigma-Aldrich, USA) and mouse monoclonal anti-KLRC3 (Sigma-Aldrich, USA) were used.

Techniques: Microarray, Binding Assay

Journal: BMC Cancer

Article Title: Regulation of KLRC and Ceacam gene expression by miR-141 supports cell proliferation and metastasis in cervical cancer cells

doi: 10.1186/s12885-024-12794-6

Figure Lengend Snippet: The relationship between miR-141 expression and KLRC gene expression profile of in HeLa cells. A Quantification of steady-state miR-141 in transfected HeLa cells compared with control-transfected and nontreated cells using qRT-PCR. B Relative gene expression of KLRC1 and KLRC3 in HeLa cells transfected with either specific inhibitor against miR-141 or miR-141 overexpression vector compared with control-transfected and nontreated cells using qRT-PCR. Error bars indicate the STD of three independent experiments. Student two-tailed t -test used for statistical analysis, (*) indicates P-values ≤ 0.05, and (**) indicates P ≤ 0.01. C Flow cytometric assay quantifies the kinetic proteins expression profile of KLRC1 (in red dots) and KLRC3 (in blue dots) in transfected cells compared with control-transfected and nontreated (NT) cells. D Western blot analysis reveals the protein expression level of KLRC1 and KLRC3 in transfected cells compared to control-transfected and nontreated cells. β-actin expression profile severed as an internal control. E Schematic representation of miR-141 binding site and seeding regions (SR) in KLRC1 and KLRC3 gene sequence indicated by IntaRNA program. F In HeLa cells pre-transfected with miR-141 overexpressing vector or specific inhibitor, the luciferase activities upon cotransfection with luciferase reporter constructs, pGL3-KLRC1 or pGL3-KLRC3 compared with cells cotransfected with pGL3-control vector. Error bars reveal the STD of three replicates. Student two-tailed t -test used for statistical analysis, (*) indicates P-values ≤ 0.05, and (**) indicates P ≤ 0.01

Article Snippet: For staining KLRC1 and KLRC3, the primary antibodies; rabbit polyclonal anti-KLRC1 (Sigma-Aldrich, USA) and mouse monoclonal anti-KLRC3 (Sigma-Aldrich, USA) were used.

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR, Over Expression, Plasmid Preparation, Two Tailed Test, Flow Cytometry, Western Blot, Binding Assay, Sequencing, Luciferase, Cotransfection, Construct

Journal: BMC Cancer

Article Title: Regulation of KLRC and Ceacam gene expression by miR-141 supports cell proliferation and metastasis in cervical cancer cells

doi: 10.1186/s12885-024-12794-6

Figure Lengend Snippet: Quantification analysis of miR-141, KLRC1, and KLRC3 in transfected HeLa cells

Article Snippet: For staining KLRC1 and KLRC3, the primary antibodies; rabbit polyclonal anti-KLRC1 (Sigma-Aldrich, USA) and mouse monoclonal anti-KLRC3 (Sigma-Aldrich, USA) were used.

Techniques: Transfection, Expressing, Control

Journal: BMC Cancer

Article Title: Regulation of KLRC and Ceacam gene expression by miR-141 supports cell proliferation and metastasis in cervical cancer cells

doi: 10.1186/s12885-024-12794-6

Figure Lengend Snippet: detailed of selected interaction between miR-141 and targeted genes

Article Snippet: For staining KLRC1 and KLRC3, the primary antibodies; rabbit polyclonal anti-KLRC1 (Sigma-Aldrich, USA) and mouse monoclonal anti-KLRC3 (Sigma-Aldrich, USA) were used.

Techniques:

Journal: Tissue Engineering. Part A

Article Title: Generation of Monkey Induced Pluripotent Stem Cell-Derived Cartilage Lacking Major Histocompatibility Complex Class I Molecules on the Cell Surface

doi: 10.1089/ten.tea.2021.0053

Figure Lengend Snippet: List of Antibodies

Article Snippet: Anti-NKG2A , Rabbit , 1:2000 , Ab260035 , abcam.

Techniques: Flow Cytometry, Immunohistochemical staining, Western Blot

Journal: Tissue Engineering. Part A

Article Title: Generation of Monkey Induced Pluripotent Stem Cell-Derived Cartilage Lacking Major Histocompatibility Complex Class I Molecules on the Cell Surface

doi: 10.1089/ten.tea.2021.0053

Figure Lengend Snippet: In vivo allogeneic transplantation of B2M −/− cyiPSC-derived cartilage. (A) Allogeneic B2M +/+ cyiPSC-derived cartilage ( top row ) and B2M −/− cyiPSC-derived cartilage ( bottom row ) were transplanted into osteochondral defects created in the joint surface of the distal femur in the knee joints of monkeys. The transplanted sites were analyzed histologically 4 weeks after the transplantation. Left , Safranin O-fast green -iron hematoxylin staining. Middle , HE staining. Right , magnification of the boxed regions in the middle panels . (B) Immunohistochemical analysis of the semiserial sections in (A) using anti-CD3 antibody ( top two rows ) and anti NKG2A antibody ( bottom two rows ). Magnification of the boxed regions in the third column is shown in the fourth column . Scale bars, 100 μm. HE, hematoxylin and eosin.

Article Snippet: Anti-NKG2A , Rabbit , 1:2000 , Ab260035 , abcam.

Techniques: In Vivo, Transplantation Assay, Derivative Assay, Staining, Immunohistochemical staining