Interferon-γ and IL-5 associated cell-mediated immune responses to HPV16 E2 and E6 distinguish between persistent oral HPV16 infections and noninfected mucosa

Abstract

Objectives: Natural history of human papillomavirus (HPV) infection in the head and neck region is poorly understood, and their impact on collective HPV-specific immunity is not known.

Materials and methods: In this study, we have performed a systematic analysis of HPV16-specific cell-mediated immunity (CMI) in 21 women with known oral and genital HPV DNA status and HPV serology (Ab) based on 6-year follow-up data. These women being a subgroup from the Finnish Family HPV Study were recalled for blood sampling to be tested for their CMI-responses to HPV16 E2, E6, and E7 peptides.

Results: The results showed that HPV16 E2-specific lymphocyte proliferation was more prevalent in women who tested HPV16 DNA negative in oral mucosa and were either HPV16 seropositive or negative than in HPV16 DNA+/Ab+ women (p = 0.046 and p = 0.035). In addition, the HPV16 DNA-/Ab- women most often displayed E6-specific proliferation (p = 0.020). Proportional cytokine profiles indicated that oral HPV16-negative women were characterized by prominent IFN-γ and IL-5 secretion not found in women with persisting oral HPV16 (p = 0.014 and p = 0.040, respectively).

Conclusions: Our results indicate that the naturally arising immune response induced by oral HPV infections displays a mixed Th1/Th2/Th17 cytokine profile while women with persisting oral HPV16 might have an impaired HPV16-specific CMI, shifted partly toward a Th2 profile, similarly as seen earlier among patients with high-grade genital HPV lesions. Thus, the lack of HPV 16 E2 and E6 specific T memory cells and Th2 cytokines might also predispose women for persistent oral HPV16 infection which might be related to the risk of cancer.

Keywords: cell-mediated immunity; cytokines; human papillomavirus; oral cavity.

FIGURE 1

The figure summarizes the women's HPV‐specific genital, oral, and serology data during the 7 years of the follow‐up (FU). At the 14‐year time point of FU, the HPV genital, anal, and oral DNAs were analyzed. Pap smears were also taken. Oral examinations were conducted by a dentist

FIGURE 2

Percentages of positive lymphocyte stimulation test (LST) responses. (a) Positive LST responses as percentages after HPV16 E2.1, E2.2, E6.2, E6.3, and E6.4 peptide pool stimulation in four subgroups. In cell culture, eight replicative wells were used for every peptide pool. Each bar is presenting the positive responsiveness for specified HPV peptide pool as a proportional distribution in different study groups. (b) Positive LST responses as percentages after HPV16 E2‐, E6‐, and E7‐specific stimulation in four study groups. (c) Positive LST responses as percentages using the combined results of E2.1 and E.2 peptide pools similar as with the results of E6.2, E6.3, and E6.4 peptide pools. Subgroups of (1) HPV16 DNA+/Ab+ (n = 4) and (2) HPV16 DNA+/Ab− (n = 6) were combined to create a group of oral HPV16‐DNA‐positive women. Subgroups of (3) HPV16 DNA−/Ab+ (n = 4) and (4) HPV16 DNA−/Ab− (n = 7) were combined to create a group of oral HPV16‐DNA‐negative women

FIGURE 3

Levels of proliferative T cell responses given as stimulation indexes (SI) against peptide pools of HPV16 E2 and E6 in 21 women. The SI values against the E2.1 and E2.2 peptides were combined similarly as with E6.2, E6.3, and E6.4 peptides. HPV16 E7 peptides did not result on specific lymphocyte stimulation (LST) in any of the subgroups. Values are presented as follows: The box is outlined on the top by the third quartile, the bottom by the first quartile, and in the middle is the median. The minimum and maximum SI values of each subgroup are marked with the whiskers. (a) SI values of four subgroups studied, (b) SI values of oral HPV16 DNA‐positive and ‐negative women

FIGURE 4

Cytokine concentrations after stimulation with HPV16‐specific peptides displayed in two or four subgroups. (a) The pooled cytokine concentrations after stimulation of HPV16 E2.1, E2.2, E6.2, E6.3, and E6.4 peptides in the four subgroups studied. Each cytokine was measured in eight replicative wells. The number of women in the subgroups was as follows: (1) HPV16 DNA+/Ab+ (n = 4), (2) HPV16 DNA+/Ab− (n = 6), HPV16 (3) HPV16 DNA−/Ab+ (n = 4), and (4) DNA−/Ab− (n = 7). The cytokine values are given as pg/ml and the boxes are outlined on the top by the third quartile, the bottom by the first quartile, and in the middle is the median. The minimum and maximum cytokine concentrations of each subgroup are marked with whiskers. (b) The pooled cytokine concentrations after stimulation of E2.1 and E2.2 peptides of oral HPV16‐DNA‐positive (n = 10) and ‐negative (n = 11) women, (c) the pooled cytokine concentrations after stimulation of E6.2, E6.3, and E6.4 peptides of oral HPV16‐DNA‐positive (n = 10) and ‐negative (n = 11) women

FIGURE 5

Proportional ratios of the cytokines measured in the lymphocyte stimulation test (LST)‐positive samples. In the upper part of the figure, the women were divided into four subgroups: HPV16 DNA+/Ab+ (n = 4), HPV16 DNA+/Ab− (n = 6), HPV16 DNA−/Ab+ (n = 4) and HPV16 DNA−/Ab− (n = 7). In the lower part of the figure, the smaller subgroups were combined to create two groups according to the oral HPV DNA status (oral HPV16‐DNA‐positive (n = 10) and ‐negative (n = 11) women). In each study subject, single‐cytokines were summed up from the LST‐positive samples, only. Then, the sum value of each cytokine was divided by the total cytokine sum values within the LST+ samples to obtain the cytokine‐specific ratios. Finally, the mean values of the given ratios were calculated for each subgroup