Abstract
Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280–288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαβ. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications.
Highlights
T lymphocytes are potent mediators of anti-tumor immune responses
SINGLE AMINO ACID SUBSTITUTIONS OF THE gp100 wt PEPTIDE DO NOT AFFECT T CELL FUNCTIONS EXCEPT FOR E3 TO A SUBSTITUTION, WHICH RESULTS IN A NULL LIGAND In order to study gp100 peptide requirements of various TCRmediated responses, both primary human T cells and Jurkat T cells were retrovirally transduced with T cell receptor (TCR) α and β genes that originated from the gp100/human leukocyte antigen (HLA)-A2-specific CTL clone 296, and MACsorted for high and equal levels of TCR expression
Flow cytometry with TCRVβ mAb showed that gp100 TCR expression levels were about 90% [mean fluorescence intensity (MFI): 103] and 93% (MFI: 214) for primary human T lymphocytes and Jurkat T cells, respectively
Summary
T lymphocytes are potent mediators of anti-tumor immune responses. T cell receptor (TCR) genes derived from antitumor T lymphocytes have been successfully used to redirect other, non-tumor-specific T lymphocytes to tumor cells, and have shown promising clinical activities in the treatment of tumor-bearing patients [1, 2]. Adoptive T cell therapy to tumors is based on the ability of TCRs to selectively recognize antigens, i.e., peptides that are presented by Major Histocompatibility Complex (MHC) molecules. The clinical use of TCR-engineered T lymphocytes directed against the human leukocyte antigen (HLA)-A2-restricted antigens MART-1, gp100, or NY-ESO-1 resulted in objective responses in patients with metastatic melanoma up to 45% [3, 4]. The avidity and antigen reactivity of parental T cell clones, used as a source for TCR genes, are preserved by TCR gene transfer [5,6,7]. Cytotoxic responses of TCR-engineered T cells toward a panel of gp100 peptide mutants are identical to those of parental CTL clones [7]
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