Comparison of cervical and blood T-cell responses to human papillomavirus-16 in women with human papillomavirus-associated cervical intraepithelial neoplasia

Abstract

Human papillomaviruses (HPVs) are obligate epithelial pathogens and typically cause localized mucosal infections. We therefore hypothesized that T-cell responses to HPV antigens would be greater at sites of pathology than in the blood. Focusing on HPV-16 because of its association with cervical cancer, the magnitude of HPV-specific T-cell responses at the cervix was compared with those in the peripheral blood by intracellular cytokine staining following direct ex vivo stimulation with both virus-like particles assembled from the major capsid protein L1, and the major HPV oncoprotein, E7. We show that both CD4(+) and CD8(+) T cells from the cervix responded to the HPV-16 antigens and that interferon-gamma (IFN-gamma) production was HPV type-specific. Comparing HPV-specific T-cell IFN-gamma responses at the cervix with those in the blood, we found that while CD4(+) and CD8(+) T-cell responses to L1 were significantly correlated between compartments (P = 0.02 and P = 0.05, respectively), IFN-gamma responses in both T-cell subsets were significantly greater in magnitude at the cervix than in peripheral blood (P = 0.02 and P = 0.003, respectively). In contrast, both CD4(+) and CD8(+) T-cell IFN-gamma responses to E7 were of similar magnitude in both compartments and CD8(+) responses were significantly correlated between these distinct immunological compartments (P = 0.04). We therefore show that inflammatory T-cell responses against L1 (but not E7) demonstrate clear compartmental bias and the magnitude of these responses do reflect local viral replication but that correlation of HPV-specific responses between compartments indicates their linkage.

Figure 1

Representative plots of cervical and blood mononuclear cells from a patient with CIN 2/3 stimulated directly ex vivo with HPV-16 L1 and E7 and stained for intracellular IFN-γ production. IFN-γ production by cervical (upper panel) and blood CD3⁺ cells (lower panel) either unstimulated or following stimulation with HPV-16 antigens L1 (VLPs) and E7. Each plot was gated on CD3⁺ cells and then analysed for CD8⁺ and IFN-γ production.

Figure 2

HPV-16 (a) L1- and (b) E7-specific IFN-γ production by CD4⁺ and CD8⁺ T cells from all donors following direct ex vivo stimulation. Individual donors have been grouped according to whether they were (i) actively infected with HPV-16 (DNA Positive; bottom panel) or (ii) were negative for HPV-16 DNA (top panel). Within each of these groups, individual donors have been grouped according to CIN status and serum IgG status. Each bar represents an individual's net percentage IFN-γ production by either CD4⁺ or CD8⁺ CD3⁺ T cells. Net percentage IFN-γ responses were calculated by subtracting background percentage IFN-γ production by unstimulated cells.

Figure 3

Impact of current HPV-16 infection at the cervix on inflammatory T cell responses to HPV-16 E7. Blood (a, b) and cervical (c, d) CD4⁺ and CD8⁺ T cell IFN-γ production following stimulation with HPV-16 E7 in women either having no HPV-16 DNA at the cervix (HPV-16 Neg) or with active HPV-16 infection (HPV-16 DNA Pos). Each (?) represents an individual's net percentage IFN-γ production by either CD4⁺ or CD8⁺ T cells. Net percentage IFN-γ responses were calculated by subtracting background percentage IFN-γ production by unstimulated cells. The solid lines indicate median response indices for each group. P-values were calculated using the Mann–Whitney U-test (Statistica®) and P-values = 0·05 were considered significant.

Figure 4

Correlation of cervical and blood CD4⁺ (a and c) and CD8⁺ (b and d) HPV-16 L1 and E7 specific T-cell response magnitudes. Each (•) represents an individual's net percentage IFN-γ production by either CD4⁺ or CD8⁺ T cells. Net percentage IFN-γ responses were calculated by subtracting background percentage IFN-γ production by unstimulated cells. The dotted line indicates the linear regression. The solid line has a slope = 1 and y-intercept = 0. To test for differences in response magnitudes between compartments, Wilcoxon rank (WR) tests for paired non-parametric data was used and these P-values have been suffixed with (WR). To test for correlation between compartments, the Spearman rank (SR) test was used and significant P-values have been suffixed with (SR). P-values = 0·05 were considered significant and appear in bold text.