An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT

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.

TCRs Genetically Linked to CD28 and CD3ε Do Not Mispair with Endogenous TCR Chains and Mediate Enhanced T Cell Persistence and Anti-Melanoma Activity

“…The use of TCR variants either lacking or containing an LCK-nonbinding mutant of CD28 IC domain demonstrated a hierarchy with respect to dependency on CD28 signaling, in particular LCK signaling for different T cell functions (CD107a , IFN-g , IL-2). This observed hierarchy is in line with an earlier report using a peptide-MHC ligand with compromised TCR binding (a partial agonist of the human gp100 280-288 peptide) (42). In fact, mobilization of CD107a generally requires limited T cell signaling and is presynthesized and ready to be mobilized, whereas production of cytokines requires de novo synthesis; in particular, IL-2 requires strong T cell signaling and NFAT activation.…”

A Molecular Switch Abrogates Glycoprotein 100 (gp100) T-cell Receptor (TCR) Targeting of a Human Melanoma Antigen

“…Indeed, even conservative peptide substitutions can have unexpected consequences for T-cell recognition due to knock-on structural changes in the HLA-bound peptide. Our findings provide a molecular explanation for the sensitivity to substitutions at gp100 280–288 peptide residue Glu 3 ( 16 , 17 ) and represent the first example of the structural mechanisms underlying T-cell recognition of this important therapeutic target for melanoma.…”

“…The CD8 + T-cell responses directed against gp100 280–288 have been shown to be polyclonal in nature ( 16 , 17 ). Along with the two TCRs under investigation here, the sequences of a further two TCRs have been published, demonstrating diverse gene usage and different CDR3 loop sequences ( Table 1 ).…”

“…Among these, the heteroclitic version of gp100 280–288 (in which a valine replaces alanine at anchor position 9 to improve pHLA stability ( 14 )) enhanced the induction of melanoma-reactive cytotoxic T lymphocytes in vitro and has been successfully used in clinical trials ( 15 ). Another heteroclitic form of gp100 280–288 , in which peptide residue Glu 3 was substituted to Ala, abrogated recognition by a polyclonal population of gp100 280–288 -specific T-cells ( 16 , 17 ). Thus, a more complete understanding of the molecular mechanisms underlying gp100 280–288 targeting by specific TCRs is needed to direct the design of improved altered peptide ligands.…”

Peptide Dose and/or Structure in Vaccines as a Determinant of T Cell Responses

“…Partial agonists are variant peptides which elicit a subset of the T cell functions generated by the wild type peptide [ 70 , 71 ]. Using a panel of gp100 substituted peptides, an altered peptide capable of inducing cytotoxic function but limited cytokine secretion from responding T cells was identified [ 72 ]. Consequently, APLs which alter the function of T cells may be useful in autoimmune disease settings or diseases dependent on a Th1/Th2 balance where silencing of selected T cell functions could be beneficial.…”