Journal: Tissue antigens

Article Title: Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

doi: 10.1111/tan.12095

Figure Lengend Snippet: (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars). The allotypes shown are those common to both beadsets.

Article Snippet: The specific reference as listed in the current publication is noted to the right. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars).

Techniques: Binding Assay

Journal: Tissue antigens

Article Title: Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

doi: 10.1111/tan.12095

Figure Lengend Snippet: (A) Binding of MA2.1 (1μg/ml) to beads coated with HLA class I allotypes from the One Lambda LabScreen (left panel) and Gen-Probe LifeCodes (right panel) beadsets. (B) Alignment of HLA class I allotypes showing selected residues in the α1 and α2 domains. Residues from allotypes that form the epitope recognized by MA2.1 are shaded in grey. (C) Space-filling model of the binding surface of HLA-A*02 (grey) with associated peptide (cyan). Residues highlighted in yellow fall within the footprint recognized by MA2.1. Residues 62–65 are critical for formation of the epitope recognized by MA2.1 and are highlighted in red.

Article Snippet: The specific reference as listed in the current publication is noted to the right. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars).

Techniques: Binding Assay

Journal: Tissue antigens

Article Title: Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

doi: 10.1111/tan.12095

Figure Lengend Snippet: (A) Binding of PA2.1 (1μg/ml) and BB7.2 (1μg/ml) to beads coated with HLA class I allotypes from the One Lambda LabScreen (left panel) and Gen-Probe LifeCodes (right panel) beadsets. (B) Binding of PA2.1 (50μg/ml) and BB7.2 (50μg/ml) to beads coated with HLA class I allotypes from the One Lambda LabScreen (left panel) and Gen-Probe LifeCodes (right panel) beadsets. (C) Alignment of HLA class I allotypes showing selected residues in the α2 domain. Residues from allotypes that form the epitope recognized by PA2.1 and BB7.2 are shaded in grey. (D) Space-filling model of HLA-A*02 (grey) with associated peptide (cyan). Residues highlighted in yellow fall within the footprint recognized by PA2.1 and BB7.2. Tryptophan at position 107 is considered critical for formation of the epitope recognized by PA2.1 and BB7.2 and is highlighted in red.

Article Snippet: The specific reference as listed in the current publication is noted to the right. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars).

Techniques: Binding Assay

Journal: Tissue antigens

Article Title: Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

doi: 10.1111/tan.12095

Figure Lengend Snippet: (A) Binding of BB7.1 (1μg/ml) to beads coated with HLA class I allotypes from the One Lambda LabScreen (left panel) and Gen-Probe LifeCodes (right panel) beadsets. (B) Alignment of HLA class I allotypes showing selected residues in the α1 and α2 domains. Residues from allotypes that form the epitope recognized by BB7.1 are shaded in grey. (C) Space-filling model of the binding surface of HLA-B*07 (grey) with associated peptide (cyan). Residues highlighted in yellow fall within the footprint recognized by BB7.1. Residues 63–71 in the a1 domain and position 131 in the α2 domain are critical for formation of the epitope recognized by BB7.1 and are highlighted in red.

Article Snippet: The specific reference as listed in the current publication is noted to the right. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Acquired IFNγ resistance impairs anti-tumor immunity and gives rise to T-cell-resistant melanoma lesions

doi: 10.1038/ncomms15440

Figure Lengend Snippet: ( a ) Clinical history of patient Ma-Mel-36. Vertical line, time axis; left, therapeutic regimens; right, primary tumour (PT)/metastases development; arrow indicates cell line established from metastasis Ma-Mel-36; grey box, stage IV disease. ( b ) IFNγ-sensitive Ma-Mel-36_sens and IFNγ-resistant Ma-Mel-36_res cells sorted from IFNγ-treated (48 h) Ma-Mel-36_bulk cells based on their HLA-DR expression profile. Surface expression of indicated proteins measured by flow cytometry. Representative data from n =3 independent experiments. ( c ) JAK1 mutation defined by targeted sequencing on DNA from Ma-Mel-36_bulk and Ma-Mel-36_res cells. Plots of aligned sequencing reads in the location where the JAK1 c.843C>A, p.Y281* mutation was identified, arrows highlight mutation or corresponding wild-type (WT) site. Number of sequencing reads notated on the left; %, frequency of mutation in reads. ( d ) Melanoma cells analysed by western blot for expression of STAT1, pSTAT1, IRF1 and HLA class I heavy chains after IFNγ treatment (48 h); GAPDH, loading control. Representative data from n =2 independent experiments. ( e ) Ma-Mel-36_sens (S) and Ma-Mel-36_res (R) cells analysed for protein expression after IFNα and IFNγ treatment (48 h). Representative data from n =2 independent experiments. ( f ) SNP results given as allelic distribution of chromosome 1p shown for DNA obtained from Ma-Mel-36_bulk, Ma-Mel-36_sens, Ma-Mel-36_res and autologous Epstein-Barr virus-transformed B cells as a control. Loss of one chromosomal allele in the region 1p36.3-1p13.1 (Chr.1:854,277-116,804,754; hg19) in all Ma-Mel-36 cell populations. Dashed line indicates JAK1 location at Chr.1p31.3. ( g ) IFNγ release by autologous CD8 + T cells in the presence of IFNγ-treated (24 h) Ma-Mel-36 cell populations measured by ELISpot assay. Mean values (+s.e.m.) from n =2 measurements. ( h ) Real-time proliferation of Ma-Mel-36 cell populations in the presence/absence of IFNγ. Bold grey vertical lines indicate addition of IFNγ. Representative data from n =3 independent experiments. ( i ) IFNγ-induced (7 days) apoptosis in Ma-Mel-36 cell populations determined by AnnexinV/PI staining. Percentage of early (AnnexinV+/PI−) and late apoptotic (AnnexinV+/PI+) cells depicted. Mean values (+s.e.m.) from n =3 independent experiments. Only statistical significant differences defined by paired Student's t -test are indicated, * P <0.05.

Article Snippet: Serial cryostat tissue sections were stained with antibodies specific for HLA-DR,-DP,-DQ, kindly provided by S. Ferrone , HMB-45 (Dako), HLA class I antigen complexes (W6/32; Dianova) in combination with a Polymer Kit containing an AP-coupled secondary antibody (ZytoChem-Plus AP Polymer Kit, Zytomed).

Techniques: Expressing, Flow Cytometry, Mutagenesis, Sequencing, Western Blot, Transformation Assay, Enzyme-linked Immunospot, Staining

Journal: Nature Communications

Article Title: Acquired IFNγ resistance impairs anti-tumor immunity and gives rise to T-cell-resistant melanoma lesions

doi: 10.1038/ncomms15440

Figure Lengend Snippet: ( a ) Clinical history of patient Ma-Mel-54. Vertical line, time axis; left, therapeutic regimens; right, primary tumour (PT)/metastases development; arrows indicate cell lines established from metastases Ma-Mel-54a and Ma-Mel-54b; grey box, stage IV disease. ( b ) Mutations defined by targeted sequencing on DNA from melanoma cells and autologous blood cells as wild-type (WT) control (ctrl). Plots of aligned sequencing reads in the location where the JAK2 c.2876A>C, p.Q959P mutation was identified. WT sequences shown on the bottom, arrows highlight mutation sites. Number of sequencing reads notated on the left; %, frequency of mutations in reads. ( c ) Lysates from Ma-Mel-54a, Ma-Mel-54b and control cells (ctrl) analysed by western blot for JAK2 expression. ( d ) Lysates from IFNγ-treated (48 h) Ma-Mel-54a and Ma-Mel-54b cells analysed for expression of STAT1, pSTAT1, IRF1 and HLA class I heavy chains. ( c , d ) GAPDH, loading control. Representative data from n =2 independent experiments. ( e ) Expression of CD54, HLA class I and PD-L1 on IFNγ-treated (48 h) melanoma cells, measured by flow cytometry. Representative data from n =3 independent experiments. ( f ) SNP results given as allelic distribution of chromosome 9p shown for DNA obtained from Ma-Mel-54a, Ma-Mel-54b and autologous peripheral blood cells as normal control (germline). Loss of one chromosomal allele in region 9p24.3–p13.2 (Chr.9:203,861-37,578,327; hg19) present in both cell lines. Dashed line indicates JAK2 location at Chr.9p24.1. ( g ) Lysates from IFNγ-treated (48 h) Ma-Mel-54a-JAK2 transfectants analysed by western blot for expression of indicated proteins. ( h ) Real-time cell proliferation in the presence or absence of IFNγ. Bold grey vertical lines indicate addition of IFNγ. ( g , h ) Representative data from n =2 independent experiments. ( i ) Ma-Mel-54a and Ma-Mel-54b cells analysed for specific mRNA expression by quantitative reverse transcription–PCR. ( j ) Ma-Mel-54a-JAK2 transfectants analysed for specific mRNA expression by quantitative reverse transcription–PCR in the presence or absence of IFNγ (48 h). ( i , j ) Relative expression levels given as means (+s.e.m.) from n =2 independent experiments. ( k ) HLA class I expression on IFNγ-treated (48 h) Ma-Mel-54a cells and Ma-Mel-54a-JAK2 transfectants, measured by flow cytometry. Representative data from n =2 independent experiments.

Article Snippet: Serial cryostat tissue sections were stained with antibodies specific for HLA-DR,-DP,-DQ, kindly provided by S. Ferrone , HMB-45 (Dako), HLA class I antigen complexes (W6/32; Dianova) in combination with a Polymer Kit containing an AP-coupled secondary antibody (ZytoChem-Plus AP Polymer Kit, Zytomed).

Techniques: Sequencing, Mutagenesis, Western Blot, Expressing, Flow Cytometry

Journal: Nature Communications

Article Title: Acquired IFNγ resistance impairs anti-tumor immunity and gives rise to T-cell-resistant melanoma lesions

doi: 10.1038/ncomms15440

Figure Lengend Snippet: ( a ) Clinical history of patient Ma-Mel-61. Vertical line, time axis; left, therapeutic regimens; right, metastases development; arrows indicate cell lines established from metastases Ma-Mel-61a, Ma-Mel-61b, Ma-Mel-61c, Ma-Mel-61e, Ma-Mel-61g and Ma-Mel-61h; grey box, stage IV disease. ( b ) Surface expression of CD54, HLA class I, and PD-L1 on IFNγ-treated (48 h) Ma-Mel-61b and Ma-Mel-61g cells, measured by flow cytometry. Representative data from n =3 independent experiments. ( c ) Mutation defined by targeted sequencing on DNA from Ma-Mel-61g cells. Plots of aligned sequencing reads in the location where the JAK1 c.1798G>T, p.G600W mutation was identified. Arrow highlights mutation site in Ma-Mel-61g or corresponding wild-type site (WT) in Ma-Mel-61b cells. Number of sequencing reads notated on the left; %, frequency of mutation in reads. ( d ) Lysates from Ma-Mel-61b and Ma-Mel-61g cells analysed by western blot for JAK1 expression; GAPDH, loading control. Representative data from n =3 independent experiments. ( e ) Lysates from IFNγ-treated (48 h) melanoma cells analysed by western blot for expression of STAT1, pSTAT1, IRF1 and HLA class I heavy chains; GAPDH, loading control. Representative data from n =3 independent experiments. ( f ) Lysates from IFNγ-treated (48 h) JAK1- transfected Ma-Mel-61g cells analysed for expression of the indicated proteins. Representative data from n =3 independent experiments. ( g ) IFNγ release by autologous CD8 + T cells in the presence of melanoma cells, measured by ELISpot assay. Means and s.e.m. (error bars) from n =4 independent measurement. Statistical significant differences defined by paired Student's t -test are indicated, * P <0.05. ( h ) Ma-Mel-61g and Ma-Mel-61g-JAK1 cells subjected to impedance-based real-time measurement of proliferation in the presence or absence of IFNγ. Addition of IFNγ indicated by bold grey vertical lines. Representative data from n =3 independent experiments. ( i ) SNP results given as allelic distribution of chromosome 1p shown for DNA obtained from the different Ma-Mel-61 cell lines and autologous blood cells as normal control (germline). Loss of one chromosomal allele in the region 1p34.3–1p12 (Chr.1:40,061,699-118,932,325; hg19) present in all cell lines. Dashed line indicates location of JAK1 at Chr.1p31.3.

Article Snippet: Serial cryostat tissue sections were stained with antibodies specific for HLA-DR,-DP,-DQ, kindly provided by S. Ferrone , HMB-45 (Dako), HLA class I antigen complexes (W6/32; Dianova) in combination with a Polymer Kit containing an AP-coupled secondary antibody (ZytoChem-Plus AP Polymer Kit, Zytomed).

Techniques: Expressing, Flow Cytometry, Mutagenesis, Sequencing, Western Blot, Transfection, Enzyme-linked Immunospot

Journal: Nature Communications

Article Title: Acquired IFNγ resistance impairs anti-tumor immunity and gives rise to T-cell-resistant melanoma lesions

doi: 10.1038/ncomms15440

Figure Lengend Snippet: ( a ) Mutation defined by targeted sequencing on DNA from Ma-Mel-61h cells and autologous blood cells as wild-type (WT) control (ctrl). Plots of aligned sequencing reads in the location where the JAK1 c.1798G>T, p.G600W mutation was identified. WT sequence shown on the bottom, arrow highlights mutation or corresponding wild-type (WT) site. Number of sequencing reads notated on the left; %, frequency of mutations in reads. ( b ) Lysates from IFNγ-treated (48 h) Ma-Mel-61b, Ma-Mel-61g and Ma-Mel-61h cells analysed by western blot for expression of IRF1 and HLA class I heavy chains; GAPDH, loading control. Representative data from n =3 independent experiments. ( c ) HLA class I and CD54 surface expression on IFNγ-treated (48 h) Ma-Mel-61g and Ma-Mel-61h cells, measured by flow cytometry. Representative data from n =3 independent experiments. ( d ) Immunohistochemical staining of serial cryostat tissue sections from metastasis Ma-Mel-61g for melanoma marker HMB45 and HLA class I. ( e ) Ma-Mel-61h and Ma-Mel-61g cells, transfected with expression plasmids encoding wild-type JAK1 or mutant JAK1-G600W, analysed for specific mRNA expression by quantitative reverse transcription–PCR in the presence of absence or IFNγ (48 h). Ma-Mel-61b cells served as a control (ctrl). Relative expression levels given as means (+s.e.m.) from n =2 independent experiments. ( f ) HLA class I surface expression on IFNγ-treated (48 h) Ma-Mel-61h-JAK1 and Ma-Mel-61h-JAK1-G600W transfectants, measured by flow cytometry. Representative data from n =2 independent experiments. ( g ) Ma-Mel-61h and Ma-Mel-61g cells, transfected with expression plasmids encoding wild-type JAK1 (WT) or mutant JAK1-G600W (M), analysed for recognition by autologous CD8 + T cells in the presence or absence of IFNγ (48 h). T-cell activation measured as IFNγ release by ELISpot assay. Representative data from n =2 independent experiments.

Article Snippet: Serial cryostat tissue sections were stained with antibodies specific for HLA-DR,-DP,-DQ, kindly provided by S. Ferrone , HMB-45 (Dako), HLA class I antigen complexes (W6/32; Dianova) in combination with a Polymer Kit containing an AP-coupled secondary antibody (ZytoChem-Plus AP Polymer Kit, Zytomed).

Techniques: Mutagenesis, Sequencing, Western Blot, Expressing, Flow Cytometry, Immunohistochemical staining, Staining, Marker, Transfection, Activation Assay, Enzyme-linked Immunospot

Journal: Cell Reports Medicine

Article Title: Neoantigen-targeted dendritic cell vaccination in lung cancer patients induces long-lived T cells exhibiting the full differentiation spectrum

doi: 10.1016/j.xcrm.2024.101516

Figure Lengend Snippet:

Article Snippet: W6/32 antibody was purified from hybridoma cell (#HB-95, ATCC) supernatant as recommended by the cell line provider.

Techniques: Derivative Assay, Recombinant, Protease Inhibitor, Cell Isolation, Multiplexing, Sample Prep, Amplification, RNA Sequencing, Immunopeptidomics, Software, Variant Assay, Sequencing, Retroviral, Chromatography, Transferring

Journal: Scientific Reports

Article Title: HLA-DR cancer cells expression correlates with T cell infiltration and is enriched in lung adenocarcinoma with indolent behavior

doi: 10.1038/s41598-021-93807-3

Figure Lengend Snippet: Mass cytometry antibody panel for lung adenocarcinoma.

Article Snippet: HLA-ABC , 144-Nd , Surface , W6/32 , Fluidigm , 3144017B.

Techniques: Mass Cytometry