CD4⁺ T cell immunity against the human papillomavirus‐18 E6 transforming protein in healthy donors: identification of promiscuous naturally processed epitopes

Abstract

Infection by 'high-risk' human papillomaviruses (HPV) is associated with the development of neoplastic lesions. HPV-18 is responsible for a very aggressive form of cancer and poor survival. As for other HPV types, immune surveillance has probably a role in the control of the infection. However, very little is known on HPV-18 immunogenicity. CD4 + T cells from 16 healthy donors were tested ex vivo for reactivity to synthetic peptides corresponding to 3 sequences on the HPV-18 E6 transforming protein predicted by bioinformatics as promiscuous HLA-DR ligands, and to the recombinant E6 protein. We found 3 donors with CD4 + T cells that specifically proliferated in the presence of HPV-18 E6 antigens and produced IFN-c in the presence of the E6 protein. We then propagated CD4 + T cell lines and clones from the responsive subjects to better characterize the recognized sequences. We show that E6 52-66 and E6 97-111 are indeed promiscuous and, most importantly, they contain naturally processed epitopes. Collectively, our data indicate that healthy donors may develop spontaneous CD4 immunity against HPV-18 E6 epitopes, thus strongly suggesting the potential for this protein to elicit in the host a natural productive immune response.

IFN-γ Produced by Human Papilloma Virus-18 E6-Specific CD4+ T Cells Predicts the Clinical Outcome after Surgery in Patients with High-Grade Cervical Lesions

“…By in vitro studies, we previously identified promiscuous HPV-18 E6 CD4 epitopes recognized by 18% of healthy donors (15). Similar frequencies were found in another study (16), in which no reactivity in cervical cancer patients was found.…”

“…17. promiscuous HLA-DR binders, as previously described (15). Peptides corresponding to selected sequences were synthesized by the stepwise solidphase method as in Ref.…”

“…Normal donors and patients, irrespective of the HPV type carried, were tested for the presence of CD4 ϩ T cells against the previously described HPV-18 E6 sequences (15). Naive CD4 ϩ T cells purified from 20 cord bloods were also tested to verify whether the culture system could induce in vitro priming.…”

Endosomal Proteases Influence the Repertoire of MAGE-A3 Epitopes RecognizedIn vivoby CD4+ T Cells

“…CD4 + T cells were purified from total PBMCs and CBMCs by magnetic separation (Miltenyi Biotech) and cultured as described previously (27,28). Briefly, CD4 + T cells (5 Â 10 4 per well) were cultured in X-VIVO 15 medium (Biowhittaker) supplemented with 3% heatinactivated normal human serum (NHS), penicillin (100 units/mL), and streptomycin (50 Ag/mL; Biowhittaker; tissue culture medium) in the presence of irradiated CD4 + -depleted PBMCs as antigen-presenting cells (APC) at 1:3 ratio in 96-well plates in six replicates for each condition.…”

Identification of Novel Subdominant Epitopes on the Carcinoembryonic Antigen Recognized by CD4+ T Cells of Lung Cancer Patients

“…ϩ -depleted PBMC as APC at a 1:3 ratio in 96-well plates in six replicates for each condition as described elsewhere (29). Stimuli were PHA (1 g/ml), as a positive control, CD4…”