Journal: Journal of Translational Medicine

Article Title: Cancer-testis antigen SLLP1 represents a promising target for the immunotherapy of multiple myeloma

doi: 10.1186/s12967-015-0562-5

Figure Lengend Snippet: SLLP1 is specifically expressed by myeloma cells. a RNA expression of SLLP1 was analyzed by RT-PCR in 11 different myeloma cell lines ( upper row ) and bone marrow samples from 11 healthy donors ( lower row ). Human testis was used as a positive control and housekeeping gene GAPDH served as an internal loading control. b Expression of SLLP1 in 5 myeloma cell lines, chronic myeloid leukemia cell line K562, 293 cells, 293 cells transfected with an SLLP1 expression plasmid and human testis lysate was confirmed by western blot analysis. Beta actin (ACTB) was used as an internal control. c Myeloma cell lines (n = 6) were analyzed for SLLP1 expression on a single-cell level applying cytoplasmic staining followed by flow cytometry. Grey areas indicate staining intensity with an irrelevant isotype control, black areas show staining with anti-SLLP1 antibody.

Article Snippet: Primers for qualitative PCR amplification of SLLP1 cDNA (Forward: 5′-AAGCTCTACGGTCGTTGTGAACTG-3′; Reverse: 5′-CTAGAAGTCACAGCCATCCACCCA-3′) and the cDNA for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Forward: 5′-TGATGACATCAAGAAGGTGG-3′; Reverse: 5′-TTTCTTACTCCTTGGAGGCC-3′) were obtained from MWG Biotech (Ebersberg, Germany).

Techniques: RNA Expression, Reverse Transcription Polymerase Chain Reaction, Positive Control, Expressing, Transfection, Plasmid Preparation, Western Blot, Staining, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: Cancer-testis antigen SLLP1 represents a promising target for the immunotherapy of multiple myeloma

doi: 10.1186/s12967-015-0562-5

Figure Lengend Snippet: Patient characteristics and association with SLLP1 expression

Article Snippet: Primers for qualitative PCR amplification of SLLP1 cDNA (Forward: 5′-AAGCTCTACGGTCGTTGTGAACTG-3′; Reverse: 5′-CTAGAAGTCACAGCCATCCACCCA-3′) and the cDNA for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Forward: 5′-TGATGACATCAAGAAGGTGG-3′; Reverse: 5′-TTTCTTACTCCTTGGAGGCC-3′) were obtained from MWG Biotech (Ebersberg, Germany).

Techniques:

Journal: Journal of Translational Medicine

Article Title: Cancer-testis antigen SLLP1 represents a promising target for the immunotherapy of multiple myeloma

doi: 10.1186/s12967-015-0562-5

Figure Lengend Snippet: Sample characteristics and association with SLLP1 expression

Article Snippet: Primers for qualitative PCR amplification of SLLP1 cDNA (Forward: 5′-AAGCTCTACGGTCGTTGTGAACTG-3′; Reverse: 5′-CTAGAAGTCACAGCCATCCACCCA-3′) and the cDNA for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Forward: 5′-TGATGACATCAAGAAGGTGG-3′; Reverse: 5′-TTTCTTACTCCTTGGAGGCC-3′) were obtained from MWG Biotech (Ebersberg, Germany).

Techniques:

Journal: Journal of Translational Medicine

Article Title: Cancer-testis antigen SLLP1 represents a promising target for the immunotherapy of multiple myeloma

doi: 10.1186/s12967-015-0562-5

Figure Lengend Snippet: Expression of SLLP1 correlates with a worse prognosis in patients treated with alloSCT. A total of 84 patients who had received alloSCT were divided into to two groups according to the SLLP1 expression status in their bone marrow as determined by RT-PCR. Curves represent Kaplan–Meier estimates of progression-free survival ( a ) and overall survival ( b ) from application of alloSCT until end of observation.

Article Snippet: Primers for qualitative PCR amplification of SLLP1 cDNA (Forward: 5′-AAGCTCTACGGTCGTTGTGAACTG-3′; Reverse: 5′-CTAGAAGTCACAGCCATCCACCCA-3′) and the cDNA for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Forward: 5′-TGATGACATCAAGAAGGTGG-3′; Reverse: 5′-TTTCTTACTCCTTGGAGGCC-3′) were obtained from MWG Biotech (Ebersberg, Germany).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: Cancer-testis antigen SLLP1 represents a promising target for the immunotherapy of multiple myeloma

doi: 10.1186/s12967-015-0562-5

Figure Lengend Snippet: Myeloma patients occasionally develop spontaneous IgG antibody responses against SLLP1. Based on the unavailability of recombinant full-length SLLP1 protein with a sufficient quality, we decided to use SLLP1 20-mer peptides overlapping by 10 amino acids for the detection of spontaneous anti-SLLP1 IgG antibody responses. a For the ELISA assays, plates were coated with two pools of peptides containing SLLP1 peptides 1–10 (pool SLLP1-1; left ) and 11–21 (pool SLLP1-2; right ), respectively, Dots indicate intensity of the signal as measured by an absorbance reader. b Target epitopes of the SLLP1-specific antibody response of MM patient UKE-164 were identified using single overlapping 20-mer peptides spanning the complete sequence of SLLP1 in an ELISA assay. Recombinant influenza nucleoprotein (NP) and tetanus toxoid (TT) were used as positive controls, recombinant SSX-2 protein served as an irrelevant control protein. Bars indicate intensity (mean + SEM) of the signal against the given epitope. All results were validated by three independent experiments.

Article Snippet: Primers for qualitative PCR amplification of SLLP1 cDNA (Forward: 5′-AAGCTCTACGGTCGTTGTGAACTG-3′; Reverse: 5′-CTAGAAGTCACAGCCATCCACCCA-3′) and the cDNA for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Forward: 5′-TGATGACATCAAGAAGGTGG-3′; Reverse: 5′-TTTCTTACTCCTTGGAGGCC-3′) were obtained from MWG Biotech (Ebersberg, Germany).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Microplate Reader Absorbance Measurement, Sequencing

Journal: Journal of Translational Medicine

Article Title: Cancer-testis antigen SLLP1 represents a promising target for the immunotherapy of multiple myeloma

doi: 10.1186/s12967-015-0562-5

Figure Lengend Snippet: Induction of T cell specificity against multiple SLLP1 peptide epitopes. SLLP1-reactive T cell cultures were generated from PBMC from three healthy donors: a UKE-1, b UKE-2, and c UKE-3. Each plot represents an SLLP1-specific T cell line (TCL). IFN-γ secretion was measured in ELISPOT assay after restimulation with T-APC loaded with single peptides. T cells stimulated with unloaded T-APC were used as background controls (no peptide). Bars indicate the number of IFN-γ spots per 25,000 T cells (mean + SEM). Black bars indicate significant reactivity above the background value + 3STD.

Article Snippet: Primers for qualitative PCR amplification of SLLP1 cDNA (Forward: 5′-AAGCTCTACGGTCGTTGTGAACTG-3′; Reverse: 5′-CTAGAAGTCACAGCCATCCACCCA-3′) and the cDNA for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Forward: 5′-TGATGACATCAAGAAGGTGG-3′; Reverse: 5′-TTTCTTACTCCTTGGAGGCC-3′) were obtained from MWG Biotech (Ebersberg, Germany).

Techniques: Generated, Enzyme-linked Immunospot

Journal: Journal of Translational Medicine

Article Title: Cancer-testis antigen SLLP1 represents a promising target for the immunotherapy of multiple myeloma

doi: 10.1186/s12967-015-0562-5

Figure Lengend Snippet: Phenotype of SLLP1-specific T cells. a T cell lines were restimulated with their cognate SLLP1 31–50 peptide or irrelevant control peptide and secretion of IFN-γ was measured on the cell surface by flow cytometry. b T cell clone number 1 (TCC1) was stained with an antibody cocktail containing three different TRBV antibodies (PE, FITC, PE/FITC). The gray dot plot represents the isotype control. c SLLP-1-specific T cell clones analyzed phenotypically using staining with anti-CD4 and anti-CD8 antibodies followed by flow cytometry.

Article Snippet: Primers for qualitative PCR amplification of SLLP1 cDNA (Forward: 5′-AAGCTCTACGGTCGTTGTGAACTG-3′; Reverse: 5′-CTAGAAGTCACAGCCATCCACCCA-3′) and the cDNA for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Forward: 5′-TGATGACATCAAGAAGGTGG-3′; Reverse: 5′-TTTCTTACTCCTTGGAGGCC-3′) were obtained from MWG Biotech (Ebersberg, Germany).

Techniques: Flow Cytometry, Staining, Clone Assay

Journal: Journal of Translational Medicine

Article Title: Cancer-testis antigen SLLP1 represents a promising target for the immunotherapy of multiple myeloma

doi: 10.1186/s12967-015-0562-5

Figure Lengend Snippet: Sequencing of SLLP1-specific T cell receptors

Article Snippet: Primers for qualitative PCR amplification of SLLP1 cDNA (Forward: 5′-AAGCTCTACGGTCGTTGTGAACTG-3′; Reverse: 5′-CTAGAAGTCACAGCCATCCACCCA-3′) and the cDNA for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Forward: 5′-TGATGACATCAAGAAGGTGG-3′; Reverse: 5′-TTTCTTACTCCTTGGAGGCC-3′) were obtained from MWG Biotech (Ebersberg, Germany).

Techniques: Sequencing