Antigen-Specific T Cells for Melanoma Researchers: gp100
Our scientists were among the first to develop custom antigen-specific T cells for research use and have created them against a diverse range of targets, including viral, tumor, and self-antigens.
We’re now developing the next generation of antigen-specific T cells to target some of today’s most prevalent and challenging diseases. More on that below. But first, a primer on antigen-specific T cells.
The Role of Antigen-Specific T Cells in Disease Research and Treatment
T cells are a major player in the immune response in various diseases, including cancer and pathogenic infections, and are mediators in inflammation and autoimmunity. A large body of research has been conducted over the past few decades to manipulate T cell responses, leading to many clinical strategies including cancer vaccines, T cell adoptive therapy, CAR-T therapy, immune-oncology, and the use of regulatory T cells for autoimmune diseases.
Antigen-specific T cells are an important tool in T cell biology research and potency assays. Investigators can isolate them directly from the disease site (e.g., tumor-infiltrating lymphocytes (TIL) or inflammatory lesion infiltrate populations), but these procedures often yield a limited number of cells due to low cell frequency and lack of robust isolation methods.
Alternatively, antigen-specific T cells can be generated in vitro using antigen-presenting cells pulsed with whole antigens or peptides. However, this process is often time-consuming with a low success rate.
About Ignyte Bio’s Antigen-Specific T Cells
Ignyte Bio offers antigen-specific T cells for research use you can rely on.
- Typically generated using multiple in vitro stimulations with peptide antigens
- Not immortalized or genetically modified, so they more closely mimic physiological T cells
- Specificity analyzed using the cognate peptide/MHC tetramer binding as well as interferon-gamma (IFN-γ) secretion assays.
Announcing Ignyte Bio’s Newest Antigen-Specific T Cell Line for Melanoma Research
Melanoma is one of the malignancies that have been studied intensively, particularly in terms of the immune response to tumors, and has been used as a model for the development of immunotherapy.
About the gp100 Antigen
gp100 (aka Pmel17) is a 661 amino acid melanocyte glycoprotein that is overexpressed in melanomas. Several gp100-derived peptides have been identified as targets of cytotoxic T cells in melanoma patients. One specific gp100 peptide — wild type gp100209-217 (ITDQVPFSV) and its analog, which contains a threonine to methionine substitution at position 210, gp100209-217, T210M (IMDQVPFSV) — have been used in several melanoma vaccine trials. The latter is shown to be more immunogenic resulting from increased binding to the HLA-A-02:01 molecule.
Ignyte Bio’s anti-gp100-specific T cells are generated against the analog peptide gp100209-217, T210M.
Figure 1 shows specific IFN-γ secretion when T cells are incubated with HLA-A-02:01+ target cell in the presence of gp100209-217, T210M peptide but not a control peptide. These T cells also recognize the wild type gp100209-217 in association with HLA–A-02:01 as shown in a tetramer binding study in Figure 2. These T cells specifically lyse HLA-A-02:01+ target cells in the presence of both wild type and analog gp100 peptides, as seen in Figure 3.
Figure 1. Recognition of Antigenic Peptide (gp100 209-217, 210M)) Lead to Specific Secretion of IFN-γ. Freshly thawed anti-gp100 CD8+ T cells were plated at 2 x 104 cells/well in the presence of 2 x 104 T2 cells, an HLA-A-02:01+ B-LCL (ATCC, Manassas, VA) and different concentrations of gp100209-217, T210M (IMDQVPFSV) or tyrosinase369-377 (YMDGTMSQV) control peptide in a 96-well round bottom plate. After 18-24 hours of incubation at 37oC, 5% CO2, culture supernatants were harvested and assayed for IFN-g using the Lumit™ IFN-γ Immunoassay (Promega, Madison, WI). IFN-γ concentration is plotted against gp100 and control peptide concentrations.
Figure 2. Recognition of Wild Type gp100 209-217 by T Cells Generated Against Mutant gp100 209-217, M210 Peptide Demonstrated by Peptide/MHC Tetramer Binding Analysis. Cryopreserved T cells were thawed and stained with APC-labelled HLA-A-02:01-gp100209-217 (ITDQVPFSV, gp100 Tetramer APC) or a control tetramer HLA-A-02:01-tyrosinase369-377 (YMDGTMSQV, TYR Tetramer APC). These cells were counterstained with anti-CD8 Pacific Blue reagent and analyzed by flow cytometry. Non-viable cells were excluded from the analysis using 7-AAD.
Figure 3. gp100-Specific T Cells Recognize and Kill Target Cells in the Presence of Mutant gp100 209-217, M210 and Wild Type gp100209-217 Peptides. Freshly thawed anti-gp100 CD8+ T cells were plated at 2 x 104 cells/well in the presence of 2 x 104 T2 cells, an HLA-A-02:01+ B-LCL (ATCC, Manassas, VA) and different concentrations of analog gp100209-217, T210M(IMDQVPFSV), wild type gp100209-217 (ITDQVPFSV) or WT-1 126-134 (RMFPNAPYL) control peptide in a 96-well round bottom plate. After 18-24 hours of incubation at 37oC, 5% CO2, cells in each well were stained with the viability dye 7-AAD and analyzed using flow cytometry. % Cytotoxicity (=% 7-AAD+ target cells) is plotted against the respective peptide concentrations.