Human papillomavirus 16-associated cervical intraepithelial neoplasia in humans excludes CD8 T cells from dysplastic epithelium

Abstract

High-grade cervical dysplasia caused by human papillomavirus (HPV) type 16 is a lesion that should be susceptible to an HPV-specific immune response; disease initiation and persistence is predicated on expression of two viral Ags, E6 and E7. In immune-competent subjects, at least 25% of HPV16(+) high-grade cervical dysplasia lesions undergo complete regression. However, in the peripheral blood, naturally occurring IFN-γ T cell responses to HPV E6 and E7 are weak, requiring ex vivo sensitization to detect, and are not sufficiently sensitive to predict regression. In this study, we present immunologic data directly assessing cervical lymphocytes from this cohort. We found that nearly all cervical tissue T cells express the mucosal homing receptor, α(4)β(7) surface integrin. T cells isolated from dysplastic mucosa were skewed toward a central memory phenotype compared with normal mucosal resident T cells, and dysplastic lesions expressed transcripts for CCL19 and CCL21, raising the possibility that the tissue itself sustains a response that is not detectable in the blood. Moreover, lesion regression in the study window could retrospectively be predicted at study entry by the ability of CD8(+) T cells to gain access to lesional epithelium. Vascular endothelial expression of mucosal addressin cell adhesion molecule-1, the ligand that supports entry of α(4)β(7)(+) T cells into tissues, colocalized tightly with the distribution of CD8 T cells and was not expressed in persistent dysplastic epithelium. These findings suggest that dysregulated expression of vascular adhesion molecules plays a role in immune evasion very early in the course of HPV disease.

FIGURE 1

CD8⁺ infiltrates localize to dysplastic mucosal. A, Representative CD8 immunohistochemical staining of persistent CIN2/3 adjacent to normal mucosa. CD8⁺ cells localize to dysplastic stroma compared with immediately adjacent normal tissue. ROIs are demarcated as indicated to illustrate the method used to quantitate the intensity of immunostaining (original magnification ×100). B, Representative section depicting CD8⁺ cells in normal cervical squamous mucosa distributed along the basal epithelial layer and clustered around a vessel extending into the squamous compartment (immunohistochemical staining, original magnification ×400). C, Representative section depicting CD8⁺ cell penetration into lesional epithelium in t₀ biopsy specimen, lesion regressor (immunohistochemical staining, original magnification ×400). D, Representative example of lack of CD8⁺ cells in lesional epithelium at t₀, nonregressor (immunohistochemical staining, original magnification ×400). E, Quantification of CD8⁺ infiltrates in cervical mucosa in normal (control) mucosa (n = 15) and from lesion sites at t₀ in lesions that regressed (n = 7) and lesions that did not regress (n = 20). At least 3 and up to 10 fields from each section were counted to define the density of infiltrates. F, Quantification of stromal and epithelial CD8⁺ in nonregressing lesions (n = 20), at study entry (t₀), and at the time of excision (t_wk15).

FIGURE 2

Dysplastic mucosa recruits activated memory T cells. A, Representative flow cytometry analysis of the percentage of CD3⁺CD45RO⁺ and CD3⁺CD45RA⁺ T cells in normal and CIN2/3 cervix. B, Quantification of CD45RA⁺ and CD45RO⁺CD3⁺ T cells in normal (n = 6) and CIN2/3 cervix (n = 4). C, Representative flow cytometry analysis of the percentage of CD3⁺ T cells expressing activation markers CD25 and CD69 in normal and CIN2/3 cervixes. D, Quantification of CD3⁺CD45RO⁺ T cells expressing CD25 and CD69 in normal (n = 3) and CIN2/3 (n = 3) cervixes. E, Representative flow cytometry analysis of CD3⁺CD45RO⁺ T cells expressing surface phenotype of effector memory T cells (CCR7⁻CD62L⁻) versus T_CM (CCR7⁺CD62L⁺) in normal and CIN2/3 cervixes. F, Quantification of effector memory T cells and T_CM in normal (n = 6) and CIN2/3 (n = 6) cervixes. G, Representative histologic sections of tonsil (left panels) and CIN2/3 cervix (right panels), immunostained for CD62L (top panels) and PNAd (bottom panels) demonstrate colocalization of cells expressing CD62L, and endothelial vasculature expressing PNAd (original magnification ×200). H, CCL19 and CCL21 mRNA transcripts relative to GADPH were quantified by quantitative real-time RT-PCR in normal (n = 3) and CIN2/3 (n = 4) flash-frozen cervical tissue samples.

FIGURE 3

Colpograph of CIN2/3: acetowhite epithelium (dashed red line) with characteristic patterns of neovascularization, including punctation, and mosaicism (bold arrow). Original magnification ×10.

FIGURE 4

Cervical tissue T cells express the α₄β₇ surface integrin. A, Representative flow cytometry surface phenotyping analyses of T cells isolated from normal cervixes (left panels) and CIN2/3 cervixes (right panels). B, Quantification of the percent of cervical CD3⁺ T cells expressing α₄β₇, CCR4, and CLA in normal (n = 5) and CIN2/3 (n = 4) cervixes.

FIGURE 5

Tissue expression of MAdCAM colocalizes with CD8⁺ infiltrates in CIN2/3 epithelium. A, Representative serial tissue sections of normal cervical epithelium, immunostained for CD8 (left panel) and MAdCAM-1 (right panel). Arrows identify perivascular CD8⁺ cells (left panel) and MAdCAM-1 expression in corresponding vessels (right panel) (original magnification ×400). B, Representative CIN2/3 epithelium containing CD8⁺ cell infiltrates (left panel), with MAdCAM-1 expression in a serial section, same field (right panel) (original magnification ×100). C, Representative histologic sections of CIN2/3 epithelium with very sparse CD8 infiltrates (left panel) and MAdCAM-1 expression in a serial section, same field (right panel) (original magnification ×100). D, Quantification of MAdCAM-1 immunostaining in normal cervical epithelium (n = 7), normal cervical stroma (n = 8), CIN2/3 epithelium (n = 9), and CIN2/3 stroma (n = 9). E, Within-specimen correlation of the intensity of MAdCAM-1 and CD8 expression in normal and CIN2/3 mucosa. Each symbol represents the density of MAdCAM-1 (x coordinate) and CD8 (y coordinate) in the same field. The overall correlation is 0.738; correlation in CIN2/3 epithelium is 0.8833. p = 0.0031.