Functional characterization of EBV-encoded nuclear antigen 1-specific CD4⁺ helper and regulatory T cells elicited by in vitro peptide stimulation

CD4⁺ helper and regulatory T (Treg) cells play important but opposing roles in regulating host immune responses against cancer and other diseases. However, very little is known about the antigen specificity of CD4⁺ Treg cells. Here we describe the generation of a panel of EBV-encoded nuclear antigen 1 (EBNA1)-specific CD4⁺ T-cell lines and clones that recognize naturally processed EBNA1-P_607-619 and -P _561-573 peptides in the context of HLA-DQ2 and HLA-DR11, -DR12, and -DR13 molecules, respectively. Phenotypic and functional analyses of these CD4⁺ T cells revealed that they represent EBNA1-specific CD4 ⁺ T helper as well as Treg cells. CD4⁺ Treg cells do not secrete interleukin (IL)-10 and transforming growth factor β cytokines but express CD25, the glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), and Forkhead Box P3 (Foxp3), and are capable of suppressing the proliferative responses of naïve CD4⁺ and CD8⁺ T cells to stimulation with mitogenic anti-CD3 antibody. The suppressive activity of these CD4⁺ Treg cells is mediated via cell-cell contact or in part by a cytokine-dependent manner. Importantly, these Treg cells suppress IL-2 secretion by CD4⁺ effector T cells specific for either EBNA1 or a melanoma antigen, suggesting that these CD4⁺ Treg cells induce immune suppression. These observations suggest that the success of peptide-based vaccines against EBV-associated cancer and other diseases may likely depend upon our ability to identify antigens/ peptides that preferentially activate helper T cells and/or to design strategies to regulate the balance between CD4 ⁺ helper and Treg cells.