Human T cell responses to HPV 16 E2 generated with monocyte-derived dendritic cells

Abstract

Persistent infection with human papillomavirus (HPV) type 16 has been implicated in the etiology of cervical cancer. The E2 protein is required early in viral infection and therefore may serve as a useful immune target for a vaccine aimed at prevention or therapy of premalignant lesions. Dendritic cells (DC) prepared from monocytes and pulsed with bacterially produced HPV 16 E2 C-terminus protein were used to stimulate autologous T cells over several rounds of stimulation. T cells were tested for ␥-interferon release by ELISPOT and for cytotoxic activity by 51 chromium release assays. To generate E2-expressing target cells for cytotoxicity assays, we constructed a recombinant vaccinia virus encoding HPV 16 E2, which was used to infect autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL). The results show that DC pulsed with E2 C-terminus protein induce ␥-interferon-releasing T cells as demonstrated by ELISPOT. Furthermore, we demonstrate E2-specific lysis of vaccinia-E2 infected autologous LCL by CD8؉ cytotoxic T lymphocytes (CTL). E2-specific CTL did not lyse untreated autologous LCL or LCL infected with wild-type vaccinia and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. In addition, T cells stimulated with DC in the absence of E2 failed to demonstrate lysis of vaccinia-E2-labeled targets. Phenotypically, CTL populations were CD3؉/CD8؉. These results will facilitate the study of naturally occurring T-cell responses to HPV E2 in patients with cervical intraepithelial neoplasia and the development of immunotherapeutic strategies designed to treat this and other HPV-associated diseases. © 2001 Wiley-Liss, Inc. Key words: human papillomavirus (HPV); E2 protein; dendritic cells; cytotoxic T lymphocytes (CTL); ␥-interferon ELISPOT; primary immune responses; cervical intraepithelial neoplasia (CIN); cervical cancer; vaccinesPersistent infection with oncogenic human papillomaviruses (HPV) such as HPV 16 is necessary for the development of invasive cervical carcinoma. 1-4 Natural immunity plays an important role in the control of HPV infection, 5-7 and the identification of HPV-specific immunity in patients 8 -11 has provided the impetus for the development of vaccines to both prevent and treat HPVassociated disease. Most therapeutic vaccines have focused on stimulating specific cell-mediated immunity to HPV 16 E6 and E7 proteins, which are expressed in all stages of HPV-related disease development and are therefore appropriate immune targets in high grade and malignant disease. 12,13 Other early viral proteins could also be immune targets for vaccines aimed at treating HPV-associated disease. HPV 16 E2 is a 42-kDa protein comprising 3 functional domains: a 200-amino acid amino-terminal (N-terminal) activation domain 14 and a 100-amino acid carboxy-terminal (C-terminal) DNA-binding domain, 15 joined by a flexible hinge region. 16 E2 is necessary for viral replication 17,18 and for the transcriptional control of other viral proteins, including E6 and E7. 19 In c...

T-cell responses to human papillomavirus type 16 among women with different grades of cervical neoplasia

“…Studies using whole HPV proteins to restimulate CTL responses, either in a soluble form (Nakagawa et al, 1997) or expressed by recombinant viral vectors (Nimako et al, 1997), where the whole protein is naturally processed, have been more successful. Autologous dendritic cells (DCs) presenting HPV16 peptides or whole proteins have also been used to restimulate E6-and E7-specific CTL responses from both normal donors and women with cervical carcinoma in vitro (Murakami et al, 1999;Schoell et al, 1999;Thornberg et al, 2000;Davidson et al, 2001;Liu et al, 2001;Chiriva-Internati et al, 2002;Valdespino et al, 2005), although it is not clear whether these represent primary or memory T-cell responses. Overall, the results suggest that naturally occurring HPV E6-and E7-specific CTLs do exist in patients with HPV-associated disease, although their detection may depend on the methods used to reactivate them in vitro.…”

Human papillomavirus type 16 E2- and L1-specific serological and T-cell responses in women with vulval intraepithelial neoplasia

“…IFN-c ELISPOT potentially allows the detection of low-frequency memory CD8 + cytotoxic T lymphocytes (CTLs) and CD4 + T helper 1 (Th1) cells, since both release IFN-c following re-exposure to specific antigens (Sallusto et al, 1999;van der Burg et al, 2001). Multiple restimulation of normal donor T-cell cultures with autologous E2 C terminus protein-pulsed DCs was shown to induce CTLs capable of E2-specific lysis of autologous lymphoblastoid cell lines infected with a recombinant vaccinia virus encoding HPV-16 E2 (Davidson et al, 2001). Thus, there is an E2-specific CTL repertoire available in normal individuals.…”

“…We have previously described an interferon-c (IFN-c) ELISPOT assay, which facilitates the detection of HPV-16 E2-specific memory T-cell responses to E2 C terminus protein following its processing and presentation by autologous dendritic cells (DCs) (Davidson et al, 2001). The advantage of this system is that it is not limited by the use of HLA-restricted peptides but rather allows the study of the full range of T-cell responses that may have arisen as a consequence of natural antigen presentation in vivo.…”

Therapeutic Vaccines for Cervical Cancer: Dendritic Cell-Based Immunotherapy

“…In this regard, E1, E2 and E4 are expressed during viral infection, and HPV16 E2-specific T helper responses have been reported in women with previous or present HPV16 infection [199]. HPVspecific CD8+ T cells from peripheral blood leukocytes have been generated against E1, E2 and E4 [200,201].…”