Rapid enrichment of human papillomavirus (HPV)-specific polyclonal T cell populations for adoptive immunotherapy of cervical cancer Annemieke de Jong 1,2 , Jeanette M. van der Hulst 1 , Gemma G. Kenter 3 , Jan Wouter Drijfhout 1 , Kees L. M. C. Franken 1 , Pieter Vermeij 2 , Rienk Offringa 1 , Sjoerd H. van der Burg 1 * and Cornelis J. M. Melief 1 1 Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands 2 Department of Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, The Netherlands 3 Department of Gynecology, Leiden University Medical Center, Leiden, The Netherlands The majority of cervical cancers are caused by human papilloma- virus type 16 (HPV16). Cervical cancer is associated with an ineffective host immune response against the HPV16 oncoproteins, characterized by the lack of the strong E6-specific T-helper type 1 (Th1) immunity that is generally present in healthy individuals, the presence of improperly polarized HPV16E6- and E7-specific CD4  T cells and increased numbers of regulatory T cells. There- fore, immunotherapeutic intervention is likely to require a modal- ity that deletes the regulatory T cell component and enhances the HPV16-specific Type 1 T cell response. HLA-matched allogeneic stem cell transplantation may offer such a modality, because it involves the eradication of host immune cells and enables the transfer of donor derived tumor-specific T cells to the patient. As a first step in the development of such a treatment, we evaluated the success rate of a protocol for enrichment of HPV16E6-specific CD4  T cells from healthy donor PBMC on the basis of their IFN secretion. After a short in vitro stimulation with overlapping 30 amino acid long HPV16E6 peptides, we enriched the IFN secret- ing cells by magnetic cell sorting. The obtained polyclonal CD4  T cell populations recognized distinct epitopes within HPV16E6, as well as E6 protein, processed and presented by autologous profes- sional antigen presenting cells. The described protocol proved successful in PBMC from more than half of the healthy adult blood donors. These HPV16E6-specific CD4  T cells may turn out to be an essential component of future adoptive T cell therapy for advanced cervical cancer, by orchestrating CTL dependent and independent tumoricidal mechanisms. © 2004 Wiley-Liss, Inc. Key words: cervical cancer; adoptive transfer; HPV-specific T cells Cervical cancer is associated with persistent high-risk human papillomavirus (HPV) infection. It is the first major solid tumor that has been shown to be virally induced in essentially all cases, and HPV type 16 (HPV16) is the high-risk type found most frequently in cervical cancer. 1 The HPV16 oncoproteins E6 and E7 are constitutively expressed in tumor cells and are required to maintain the malignant phenotype. 2 Therefore, these proteins are attractive targets for immunotherapy and, as such, have inspired the development of immunotherapeutic strategies for the treatment of patients with cervical cancer. 3 Advanced cervical cancer is associated with tumor-related im- munosuppression, 4 the presence of increased numbers of regula- tory T cells 5 and improperly polarized HPV-specific T cells. 6,7 Under these circumstances, active therapeutic vaccination will likely fail to establish effective immunity. An alternative approach is a passive therapeutic modality involving the adoptive transfer of tumor-specific T cells. In murine tumor models, adoptive T cell therapy has proven successful for the eradication of established solid tumors. 8,9 In patients with solid tumors, the adoptive transfer of tumor-specific T cells has mainly been applied in the treatment of metastatic melanoma. These protocols have shown a variable success rate, 10 probably due to the fact that the majority of the protocols focused on the generation of large numbers of CD8 + T cells 11 and dismissed the tumor-specific CD4 + T cell popula- tion, which forms an essential component of the anti-tumor- immunity. 12–14 In addition, the autologous setting in which these adoptive T cell transfers are carried out, involves 2 major draw- backs. First, ex vivo expanded patient derived T cell populations (especially tumor infiltrating lymphocytes) may harbor improperly polarized and regulatory T cells that can hamper the treatment efficacy. 15 Secondly, pre-existing immunoregulatory mechanisms present in advanced cervical cancer patients may prevent the effector function of adoptively transferred tumor-specific T cells. To overcome the aforementioned barriers, HLA-matched alloge- neic stem cell transplantation (allo-SCT) involving the eradication of host immune cells, including regulatory T cells, and enabling the administration of tumor-specific T cells of donor origin, may offer an alternative option. The infusion of donor lymphocytes after allo-SCT has proven relatively successful in the treatment of relapsing hematological malignancies. 16,17 In these cases, effective therapy is often based on the in vivo induction of T cells specific for minor histocompatibility antigens expressed in host-cells of hematological origin. 18 However, the incorporation of allo-SCT in the treatment of solid tumors has been less successful. 19,20 This could be due to the lack of defined tumor-specific antigens, as well as tumor-induced tolerization of the donor-derived immune sys- tem, 21,22 which may occur during immune reconstitution. Cervical cancer has the advantage over several other solid tumors that it harbors well-defined tumor-specific antigens of viral origin. The allo-SCT can serve as a therapeutic platform to administer HPV16- specific T cells derived from the peripheral blood of the healthy stem-cell donor. The adoptive transfer of HPV16E6-specific Th1- type CD4 + T cells, which are readily detected in the peripheral blood of the majority of healthy subjects, but are often lacking in HPV16-positive cervical cancer patients, 7,23 may contribute to the anti-tumor immunity. Tumor-specific CD4 + Th1 type T cells have emerged as an essential component in anti-tumor immunity, ful- filling a multifactorial role, including the activation of antigen presenting cell (APC) maturation for efficient CD8 + priming, the release of cytokines important in CD8 + T cell proliferation and differentiation, and in the recruitment of other effector cells such as eosinophils and macrophages, capable of exerting anti-tumor reactivity. 12,24 Furthermore, the anti-angiogenic activity of CD4 + Th1 type T cell-derived IFN can reportedly mediate tumor rejec- tion. 25 All of these activities orchestrated by tumor-specific CD4 + T cells are independent of MHC Class II expression on the tumor. As a first step in the development of a T cell based treatment modality, we set up a protocol for the enrichment of Th1 type HPV16E6-specific T cells from the peripheral blood of healthy blood donors. This protocol involves brief in vitro stimulation of Abbreviations: APC, antigen presenting cell; CIN, cervical intraepithe- lial neoplasia; CTL, cytotoxic T lymphocyte; CxCa, cervical carcinoma; ELISA, enzyme-linked immunosorbent assay; HPV, human papillomavi- rus; ICS, intracellular cytokine staining; IFN, interferon; MACS, magnetic cell sorting; PBMC, peripheral blood mononuclear cells; Th, T-helper. Grant sponsor: Dutch Cancer Society; Grant number: NKB:RUL 99- 2024. *Correspondence to: Dept. of Immunohematology and Blood Trans- fusion, Building 1, E3-Q, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. Fax: +31-71-5216751. E-mail: shvdburg@lumc.nl Received 17 June 2004; Accepted after revision 30 August 2004 DOI 10.1002/ijc.20721 Published online 11 November 2004 in Wiley InterScience (www. interscience.wiley.com). Int. J. Cancer: 114, 274 –282 (2005) © 2004 Wiley-Liss, Inc. Publication of the International Union Against Cancer