New Tetramer Technology Offers New Aspects in the Definition of Appropriate HPV Target Epitopes for Novel Vaccine Designs in Cervical Cancer Patients[*]

Zusammenfassung

Fragestellung: Die für die neoplastische Zelltransformation essenziellen Onkoproteine E6 und E7 von HPV-16 repräsentieren ideale Zielantigene einer virusspezifischen, MHC-Klasse-I-restringierten zellulären Immunantwort bei Zervixkarzinompatientinnen. Ziel der vorliegenden Untersuchung war es, HPV-16-E7-spezifische zytotoxische T-Zellen in unterschiedlichen biologischen Kompartimenten (periphäres Blut, Primärtumor und drainierende pelvine Lymphknoten) zu identifizieren. Dazu benutzten wir die neue Methode der Tetramer-Analyse.

Methode: Sortierte CD8 + T-Zellen (Blut, Tumor, Lymphknoten) von gesunden Probandinnen (n = 10), Patientinnen mit CIN (n = 9) und invasivem Zervixkarzinom (n = 15) wurden mittels Tetramer-Analyse hinsichtlich ihrer HPV-16-E7-Spezifität evaluiert. Die HLA- und HPV-Typisierung erfolgte durch PCR. Probandinnen und Patientinnen waren alle HLA-A2-positiv. Alle Tumorpatientinnen waren HPV-16-positiv.

Ergebnisse: 50 % der gesunden Probandinnen zeigten im peripheren Blut eine zytotoxische CD8 + Immunantwort (> 0,1 % der Gesamt-T-Zellpopulation) gegen ein definiertes HPV-16 E7_{11 - 19}-Epitop (YMLDLQPET), wohingegen lediglich bei weniger als 20 % der Patientinnen (CIN, Tumor) eine entsprechende spezifische T-Zellantwort objektiviert werden konnte. Lediglich bei einer Tumorpatientin zeigten aus Lymphknoten expandierte T-Zellen eine zytotoxische Virusspezifität.

Schlussfolgerung: Durch den Einsatz der neuen Tetramer-Technologie können wahlweise definierte tumorassoziierte Antigene bestimmt und zytotoxische T-Zellen aus allen biologischen Kompartimenten ohne In-vitro-Manipulation hinsichtlich ihrer Antigenspezifität evaluiert werden. Darüber hinaus lassen sich mit dieser Methode auch mögliche Ziel-Epitope einer effizienten Anti-HPV-Impftherapie definieren.

Abstract

Purpose: Human papillomavirus (HPV)16 E6 and E7 oncoproteins that are required for cellular transformation represent candidate targets for HPV specific and major histocompatibility complex (MHC) class-I restricted CD8 + T-cell responses in patients with cervical cancer. In the present study, we identified HPV specific T-cells in different biological compartments (peripheral blood lymphocytes [PBL], tumor tissue, tumor-draining lymphnodes) using MHC-class I tetramers containing an appropriate HPV 16 E7 peptide.

Material and Methods: FACS sorted (fluorescence activated cell sorting) CD8 + T-cells isolated from healthy blood donors (n = 10), patients with carcinoma in situ (n = 9) and invasive cervical cancer (n = 15) were obtained from PBL, tumor and pelvic nodes and evaluated for the presence of HPV 16 E7 specific effector CD3 + lymphocytes using the new tetramer technology. HPV DNA in tumor tissue was detected by PCR. All cervical cancer lesions could be identified as HPV 16 positive. PBL, tumor infiltrating lymphocytes (TIL), or T-cells from tumor-draining lymph nodes were isolated, gated on CD3 + CD8 + T-cells as indicated and tested for the frequency of HPV 16 E7 using HLA-A2 tetramer complexes. All patients included showed an HLA-A2 haplotype.

Results: 50 % of healthy individuals, but less than 20 % of patients (cervical intraepithelial neoplasia (CIN), invasive cancer) showed a specific cytotoxic CD8 + immune response to a defined HPV 16 E7_{11 - 19} epitope (YMLDLQPET) in peripheral blood. HPV-reactive cytotoxic T-cells were detectable in tumor-draining lymph nodes in only one patient.

Conclusions: The tetramer-sorting of CD8 + T-cells from different biological compartments in patients with HPV associated cancer lesions permits the determination of the target specificity of CD8 + effector T-cells without further need for in vitro manipulation. Furthermore, this new method helps to define the most appropriate target epitopes for novel vaccine designs.

HPV - Zervixkarzinom - zelluläre Immunantworten - Immuntherapie

HPV - cervical carcinoma - T-cell response - immunotherapy

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1 This manuscript partly contains published results from the Dr. med. thesis of Katia Nilges [[24]].