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

 

 Buy Article Permissions and Reprints

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.

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

Literatur

• 1 Whiteside T L. Monitoring of antigen-specific cytolytic T lymphocytes in cancer patients receiving immunotherapy.  Clin Diagnostic Lab Immunol. 2000;  7 327-332
  PubMedGoogle Scholar
• 2 Tully G, Jäger E, Maeurer M J. Detection of antigen-specific T-cells with MHC/peptide-tetramer-complexes.  Clin Appl Immunol Rev. 2002;  2 345-356
  PubMedGoogle Scholar
• 3 Pilch H, Günzel S, Schäffer U, Tanner B, Brockerhoff M, Maeurer M, Höckel M, Hommel G, Knapstein P G. The presence of HPV DNA in cervical cancer: correlation with clinico-pathological parameters and prognostic significance: 10 years' experience at the Department of Obstetrics and Gynecology of the Mainz University.  Int J Gynecol Cancer. 2001;  11 39-48
  PubMedGoogle Scholar
• 4 Zitz J, McLachlin C, Tata J, Mutter G, Crum C. Analysis of isotope labelled PCR-amplified HPV-DNA by RFLP provides optimal sensitivity and contamination detection.  Mod Pathol. 1994;  7 407-411
  PubMedGoogle Scholar
• 5 Maeurer M, Necker A, Salter R, Castelli C, Hoehn H, Karbach J, Freitag K, Neukirch C, Knuth A, Jager E. Improved detection of melanoma antigen-specific T-cells expressing low or high levels of CD8 by HLA-A2 tetramers presenting a Melan-A/MART-1 peptide analogue.  Int J Cancer. 2002;  97 64-71
  CrossrefPubMedGoogle Scholar
• 6 Hohn H, Julch M, Pilch H, Kortsik C, Tully G, Neukirch C, Freitag K, Maeurer M. Definition of the HLA-A2 restricted peptides recognized by human CD8 effector T cells by flow-assisted sorting of the CD8 + CD45RA + CD28-cell subpopulation.  Clin Exp Immunol. 2003;  131 102-110
  CrossrefPubMedGoogle Scholar
• 7 Whiteside T, Rammensee H G, Keilholz U. A sensitive ELISPOT assay for detection of CD8 + T-lymphocytes specific for HLA class I binding peptide epitopes derived from influenza proteins in the blood of healthy blood donors and melanoma patients.  Clin Cancer Res. 1997;  3 221-226
  PubMedGoogle Scholar
• 8 Altmann J D, Moss P A, Goulder P J, Barouch D H, McHeyzer-Williams M G, Bell J I, McMichael A J, Davis M M. Phenotypic analysis of antigen-specific T lymphocytes.  Science. 1996;  274 94-96
  CrossrefPubMedGoogle Scholar
• 9 Lee P P, Yee C, Savage P A, Fong L, Brockstedt D, Weber J S, Johnson D, Swetter S, Thompson J, Greenberg P D, Roederer M, Davis M M. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients.  Nat Med. 1999;  5 677-685
  PubMedGoogle Scholar
• 10 Maeurer M, Ogg C S, McMichael A J. HLA-peptide tetrameric complexes.  Curr Opin Immunol. 1998;  10 393-396
  CrossrefPubMedGoogle Scholar
• 11 Maeurer M, Chen H D, Fraire A E, Joris I, Brehm M A, Welsh R M, Selin L K. Memory CD8 + T cells in heterologous antiviral immunity and immunopathology in the lung.  Nat Immunol. 2001;  2 1067-1076
  CrossrefPubMedGoogle Scholar
• 12 Loftus D J, Ssquarcina P, Nielson M B, Geisler C, Castelli C, Odum N, Appella E. et al . Peptides derived from self-proteins as partial agonists and antagonists of human CD8 + T-cell clones reactive to melanoma/melanocyte epitome MART 1 (27 - 35).  Cancer Res. 1998;  58 2433-2439
  PubMedGoogle Scholar
• 13 Loftus D J, Castelli C, Clay T M, Squarcina P, Marincola F M, Nishimura M I. et al . Identification of epitope mimics recognized by CTL reactive to the melanoma/melanocyte-derived peptide MART1(27 - 35).  J Exp Med. 1996;  184 647-657
  CrossrefPubMedGoogle Scholar
• 14 Whelan J A, Dunbar P R, Price D A, Purbhoo M A, Lechner F, Ogg C S. Specificity of CTL interactions with peptide-MHC class I tetrameric complexes is temperature dependent.  J Immunol. 1999;  163 4342-4348
  PubMedGoogle Scholar
• 15 Rubio-Godoy V, Dutoit V, Rimoldi D, Llienard D, Lejeune F, Speiser D. et al . Discrepancy between ELISPOT IFN-gamma secretion and binding of A2/peptide multimers to TCR reveals interclonal dissociation of CTL effector functions from TCR-peptide/MHC complexes half-life.  Proc Natl Acad Sci USA. 2001;  98 10302-10307
  CrossrefPubMedGoogle Scholar
• 16 Tully G, Jäger E, Maeurer M J. Detection of antigen-specific T-cells with MHC/peptide-tetramer-complexes.  Clin Appl Immunol Rev. 2002;  2 345-356
  PubMedGoogle Scholar
• 17 Maeurer M J, Necker A, Salter R D, Castelli C, Hohn H, Karbach J. et al . Improved detection of melanoma antigen specific T cells expressing low or high levels of CD8 by HLA-A2 tetramers presenting a Melan-A/Mart-1 peptide analogue.  Int J Cancer. 2002;  97 64-71
  CrossrefPubMedGoogle Scholar
• 18 McMichael A J, Kelleher A. The arrival of HLA class II tetramers.  J Clin Invest. 1999;  104 1669-1670
  PubMedGoogle Scholar
• 19 Kotzin B L, Falta M T, Crawford F, Rosloniec E F, Bill J, Marrack P. et al . Use of soluble peptide-DR4 tetramers to detect synovial T-cells specific for cartilage antigens in patients with rheumatoid arthritis.  Proc Natl Acad Sci USA. 2000;  97 291-296
  CrossrefPubMedGoogle Scholar
• 20 Schiffmann M. Recent progress in defining the epidemiology of human papillomavirus infection and cervical neoplasia.  J Nat Cancer Inst. 1992;  84 394-398
  PubMedGoogle Scholar
• 21 Tijong Out M YTA, Schegget ter J, Burger M P, van der Vange N. Epidemiologic and mucosal immunologic aspects of HPV infection and HPV related cervical neoplasia in the lower genital female tract: a review.  Int J Gynecol Cancer. 2001;  11 9-17
  CrossrefPubMedGoogle Scholar
• 22 Ritz U, Momburg F, Pilch H, Huber C, Maeurer M J, Seliger B. Deficient expression of components of the MHC class I antigen processing machinery in human cervical cancer.  Int J Oncol. 2001;  19 1211-1220
  PubMedGoogle Scholar
• 23 Pilch H, Hohn H, Günzel S, Schäffer U, Seelig R, Seeliger B, Maeurer M, Knapstein P G. Cervical cancer cells present HPV-E7 epitope to CD4 + tumor infiltrating lymphocytes (TIL) in association with histocompatibility leucocyte antigen (HLA)DR4.  Geburtsh Frauenheilk. 1999;  59 1-6
  Article in Thieme ConnectPubMedGoogle Scholar
• 24 Nilges K, Hohn H, Pilch H, Neukirch C, Freitag C, Talbot P J, Maeurer M. HPV 16-E7 peptide directed CD8 + T-cells from patients with cervical cancer are cross-reactive to the coronavirus NS2 protein.  J Virology. 2003;  77 5464-5474
  PubMedGoogle Scholar
• 25 Oerke S, Hohn H, Zehbe I, Pilch H, Schicketanz K H, Hitzler W E, Neukirch C, Freitag K, Maeurer M J. Naturally processed and HLA-B8-presented HPV 16 E7 epitope recognized T cells from patients with cervical cancer.  Int J Cancer. 2004;  Epup ahead of print 17 Dec 2004
  PubMedGoogle Scholar
• 26 Gückel B, Meuer S, Bastert G, Wallwiener D. Einsatz tumorassoziierter Antigene in der Immuntherapie und Immundiagnostik des Mammakarzinoms.  Geburtsh Frauenheilk. 2003;  63 515-523
  Article in Thieme ConnectPubMedGoogle Scholar
• 27 Schauf B. Kongressbericht. Kongress der Mittel- und Oberrheinischen Gesellschaften für Gynäkologie und Geburtshilfe in Heidelberg.  Geburtsh Frauenheilk. 2003;  63 274-279
  Google Scholar

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

Priv.-Doz. Dr. med. Henryk Pilch Ltd. Arzt

Universitätsfrauenklinik Leipzig

Philipp-Rosenthal-Straße 55

04103 Leipzig

Email: Henryk.Pilch@medizin.uni-leipzig.de