Increase of human papillomavirus-16 E7-specific T helper type 1 response in peripheral blood of cervical cancer patients after radiotherapy

Abstract

It has been suggested that tumour cell lysis by gamma-radiation induces a tumoral antigen release eliciting an immune response. It is not clear how a specific immune response in cervical cancer patients is developed after radiotherapy. This study is an attempt to investigate the role of the human papillomavirus type 16 (HPV-16) E7-specific T helper response before and after radiotherapy. Lymphocytes were isolated from 32 cervical cancer patients before and after radiotherapy and from 16 healthy women. They were stimulated for 12 hr with autologous HPV-16 E7-pulsed monocyte-derived dendritic cells or directly with HPV-16 E7 synthetic peptides: E7(51-70), E7(65-84) and E7(79-98). The cells were stained for CD4, CD69, intracellular interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) cytokines and analysed by flow cytometry. A specific CD4(+) CD69(+) IFN-gamma(+) immune response against HPV-16 E7(79-98) peptide was observed in 10 of 14 patients (71.4%) after treatment, compared with 4 of 14 (28.5%) before radiotherapy (P = 0.039); however, this response was not associated with a successful clinical response. Before treatment, 5 of 31 patients showed a HPV-16 E7(79-98)-specific T helper type 2 (Th2) response. Interestingly, this response was significantly associated with a decrease in disease-free survival (P = 0.027). These results suggest that a Th2-type cellular response could be useful as a predictor of recurrence and poor prognosis. An increase of the HPV-specific immune response was observed after radiotherapy; however, it is not enough to control completely the disease after treatment. Our results support that the E7-specific T-cell IFN-gamma response in cervical cancer patients, rather than reflecting the host's capability of controlling tumour growth, might be an indicator for disease severity.

Figures

Figure 1

(a) Phenotype of monocyte-derived dendritic cells (DCs). The black histogram illustrates the expression of CD14 in monocytes at day 1, and the black line illustrates the expression of CD14 after 5 days. Level of expression of CD1a, CD86, HLA-DR and CD83 markers after 5 days. Isotype-matched control antibody is represented as a dotted line. Data are from a representative assay. Morphological characteristics of immature DCs (iDCs) at day 5 of differentiation. (b) Kinetics of human papillomavirus type 16 (HPV-16) E7 protein uptake and activation of mature DCs (mDCs). Indirect immunofluorescence of uptake kinetics of HPV-16 E7 protein by DCs during their maturation. The histograms illustrate the expression of CD86, HLA-DR, and CD83 markers after lipopolysaccharide (LPS) stimulus in loaded HPV-16 E7 DCs. Dotted line shows staining with isotype-matched control antibody, the grey and dark histograms show DCs after 0 hr and 24 hr treatment respectively and the dark line shows mDCs with the HPV-16 E7 protein at 48 hr. Data are from a representative assay.

Figure 2

Multiparametric flow cytometric analysis; 30 000–50 000 gated CD4+ events (R2) were acquired and analysed using cellquestpro software. The data are representative of pretreatment patients. Analysis of the percentage of human papillomavirus (HPV)-specific CD4+ T cells was performed at the R3 gate.

Figure 3

Frequencies of CD4+ CD69+ IFN-γ+ E7-specific T cells in response to human papillomavirus type 16 (HPV-16) E7 antigens in cervical cancer patients (•) and the control group (▴). The solid lines represent the mean frequency. Frequencies of non-responders were below 0·02 and they are not shown in the graph.

Figure 4

(a) Frequencies of CD4+ CD69+ IFN-γ+ or CD4+ CD69+ IL-4 E7-specific T cells before and after treatment from two patients with cervical cancer. (b) Frequencies of CD4+ CD69+ IFN-γ+ E7-specific T cells and (c) CD4+ CD69+ IL-4+ E7-specific T cells in response to E779–98 peptide in cervical cancer patients before and after treatment. The dashed lines represent the patients that showed recurrence and the dashed line ended with a square symbol represents the patient who died.

Figure 5

Disease-free survival with respect to human papillomavirus type 16 (HPV-16) E778–98-specific immune response. Kaplan–Meier curves of disease-free survival in months for responders and non-responders. (a) Th1 HPV-16 E7-specific immune response after treatment. (b) Th2 HPV-16 E7-specific immune response before treatment (P, log rank test).

Figure 6

Analysis of human leucocyte antigen (HLA) I expression in cervical tumour cells. HLA I expression was analysed by immunohistochemistry in biopsies from patients with cervical cancer. Three expression levels were determined: total loss (negative), down-regulation (low expression) and positives.