Wolfram Osen

Immune response to human papillomavirus 16 L1E7 chimeric virus-like particles: Induction of cytotoxic T cells and specific tumor protection

Expression of human papillomavirus type 16 (HPV 16) fusion proteins L1ΔCE71–55 and L1ΔCE71–60 (carboxy‐terminal deletion of L1 replaced by 55 or 60 amino‐terminal amino acids of E7) leads to formation of chimeric papillomavirus‐like particles (CVLPs). After “infection” of cells by CVLPs, the chimeric proteins can be detected in the cytosol and the endoplasmic reticulum (ER), suggesting that they are intracellularly processed via the MHC class I pathway and, therefore, able to activate cytotoxic T lymphocytes (CTLs). To investigate the cytotoxic immune response against HPV 16 L1ΔCE71–60 and L1ΔCE71–55 CVLPs, we immunized C57Bl/6 mice with various CVLP doses without adjuvant. Two weeks after immunization, spleen cells were prepared and stimulated in vitro using HPV 16 E7‐expressing transfectants of the tumor cell line RMA. In 51Cr‐release cytotoxicity assays, spleen cells of mice vaccinated with L1ΔCE71–60 CVLPs specifically lysed the RMA‐E7 transfectants as well as RMA cells loaded with the peptide E749–57, which represents an H2‐Db‐restricted CTL epitope. This demonstrates that CVLPs induce an E7‐specific CTL response in mice in the absence of an adjuvant. Furthermore, immunization with CVLPs prevented outgrowth of E7‐expressing tumor cells even if inoculation of cells was performed 2 weeks before vaccination. We conclude from our data that CVLPs show promise for therapy of HPV‐associated lesions. Int. J. Cancer 81:881–888, 1999.

Human Papillomavirus Type 16 L1 Capsomeres Induce L1-Specific Cytotoxic T Lymphocytes and Tumor Regression in C57BL/6 Mice

ABSTRACT We analyzed capsomeres of human papillomavirus type 16 (HPV16) consisting of the L1 major structural protein for their ability to trigger a cytotoxic T-cell (CTL) response. To this end, we immunized C57BL/6 mice and used the L1165-173 peptide for ex vivo restimulation of splenocytes prior to analysis (51Cr release assay and enzyme-linked immunospot assay [ELISPOT]). This peptide was identified in this study as a Db-restricted naturally processed CTL epitope by HPV16 L1 sequence analysis, major histocompatibility complex class I binding, and 51Cr release assays following immunization of C57BL/6 mice with HPV16 L1 virus-like particles (VLPs). HPV16 L1 capsomeres were obtained by purification of HPV16 L1 lacking 10 N-terminal amino acids after expression in Escherichia coli as a glutathione S-transferase fusion protein (GST-HPV16 L1ΔN10). Sedimentation analysis revealed that the majority of the purified protein consisted of pentameric capsomeres, and assembled particles were not observed in minor contaminating higher-molecular-weight material. Subcutaneous (s.c.) as well as intranasal immunization of C57BL/6 mice with HPV16 L1 capsomeres triggered an L1-specific CTL response in a dose-dependent manner as measured by ELISPOT and 51Cr release assay. Significant reduction of contaminating bacterial endotoxin (lipopolysaccharide) from the capsomere preparation did not diminish the immunogenicity. Antibody responses (serum and vaginal) were less robust under the experimental conditions employed. In addition, s.c. vaccination with HPV16 L1 capsomeres induced regression of established tumors expressing L1 determinants (C3 tumor cells). Our data demonstrate that capsomeres are potent inducers of CTL responses similar to completely assembled T=7 VLPs. This result is of potential relevance for the development of (combined prophylactic and therapeutic) HPV-specific vaccines, since capsomeres can be produced easily and also can be modified to incorporate heterologous sequences such as early HPV proteins.

Activation of p38 Mitogen-Activated Protein Kinase Drives Dendritic Cells to Become Tolerogenic in Ret Transgenic Mice Spontaneously Developing Melanoma

Purpose: The purpose of the study was to investigate signaling molecules involved in the acquisition of tolerogenic properties by dendritic cells (DC) in ret transgenic mice with spontaneous melanoma progression and to target these molecules to overcome the barrier for effective melanoma immunotherapy. Experimental Design: DC functions and expression patterns of p38 mitogen-activated protein kinase (MAPK) in DCs were evaluated in a ret transgenic murine cutaneous melanoma model, which shows high similarity to human cutaneous melanoma with respect to clinical development. In contrast to transplantation melanoma models (like B16), this model allows the study of melanoma progression under conditions of natural interactions between tumor and host cells over time. Results: We showed a strong tumor infiltration with immature DCs and a reduction in the number of mature DCs in lymphoid organs during melanoma progression. DCs from melanoma-bearing mice secreted significantly more interleukin 10 and less interleukin 12p70, and showed a decreased capacity to activate T cells compared with DCs from tumor-free animals. Observed DC dysfunction was linked to considerable activation of p38 MAPK. Inhibition of its activity in spleen DCs from tumor-bearing mice led to normalization of their cytokine secretion pattern and T-cell stimulation capacity. Conclusions: Our data show a critical role of constitutively activated p38 MAPK in the acquirement of tolerogenic pattern by DCs during melanoma progression that contributes to the suppression of antitumor T-cell immune responses. We suggest that new strategies of melanoma immunotherapy can include inhibitors of p38 MAPK activity in DCs.

Melanoma-Specific Memory T Cells Are Functionally Active in Ret Transgenic Mice without Macroscopic Tumors

We previously reported that bone marrows of breast cancer patients contained tumor antigen-specific CD8(+) T cells with central or effector memory phenotype. Using a recently developed ret transgenic mouse melanoma model, we now show that bone marrows and tumors of transgenic mice contain high frequencies of CD8(+) T cells specific for the melanoma antigen tyrosinase-related protein 2 and showing mostly effector memory phenotype. Moreover, increased numbers of bone marrow tyrosinase-related protein-2-specific effector memory CD8(+) T cells are also detected in transgenic animals older than 20 weeks with disseminated melanoma cells in the bone marrow and lymph nodes but showing no visible skin tumors and no further melanoma progression. After a short-term coincubation with dendritic cells generated from the bone marrow and pulsed with melanoma lysates, bone marrow memory T cells from mice without macroscopic melanomas produced IFN-gamma in vitro and exerted antitumor activity in vivo after adoptive transfer into melanoma-bearing mice. Our data indicate that functionally active bone marrow-derived melanoma-specific memory T cells are detectable at the phase of microscopic tumor load, suggesting that thereby they could control disseminated melanoma cells.

Specificity of human cytotoxic T lymphocytes induced by a human papillomavirus type 16 E7-derived peptide

In order to establish tumour-specific cytotoxic T lymphocyte (CTL) cell lines, T cells from a human papillomavirus (HPV) type 16-positive patient with a cervical carcinoma in situ and from a healthy volunteer were stimulated in vitro with autologous dendritic cells loaded with peptides derived from the viral transforming proteins E6 and E7 and corresponding to potential HLA-A*0201-restricted T cell epitopes. From each donor a small number of low-affinity CTL lines against the peptide E7/86-93 was obtained, which specifically lysed HLA-A*0201-expressing B-lymphocytes (cell line 721) loaded with this peptide. Cytotoxicity was also observed against two HLA-A*0201-E7-positive epithelial cell lines, the cervical carcinoma cell line CaSki and the HPV-16-immortalized foreskin-keratinocyte cell line HPK IA. However, since none of the CTL recognized both cell lines, and E7-expressing 721 transfectants were never lysed, it was concluded that the reactivity against CaSki and HPK IA cells was due to cross-reactivity on allogeneic HLA molecules rather than to E7 recognition, which emphasizes that the specificity of tumour cell lysis by peptide-induced CTL has to be interpreted with caution.

Skin melanoma development in ret transgenic mice despite the depletion of CD25+Foxp3+ regulatory T cells in lymphoid organs.

CD4+CD25+Foxp3+ regulatory T cells (Treg) known to mediate self-tolerance were also shown to contribute to tumor progression. In mouse melanoma transplantation models, Treg depletion resulted in the stimulation of antitumor immune responses and tumor eradication. To study Treg in conditions close to the clinical situation, we used a ret transgenic mouse spontaneous melanoma model, which, in contrast to transplantation models, resembles human melanoma regarding clinical development. Significantly higher numbers of Treg were found in skin tumors and metastatic lymph nodes at early stages of melanoma progression compared with more advanced stages accompanied by the elevated CCR4 expression on Treg and higher production of its ligand CCL2 in tumor lesions. Numbers of tumor infiltrating Treg inversely correlated with Treg amounts in the bone marrow, suggesting their possible recruitment to melanoma lesions from this organ. The immunosuppressive function of Treg from transgenic tumor-bearing mice was similar to that from transgenic tumor-free mice or nontransgenic littermates. Although anti-CD25 mAb injections resulted in the efficient Treg depletion from lymphoid organs of transgenic mice, melanoma development was not significantly delayed. Furthermore, the treatment of mice with macroscopical tumors also failed to inhibit tumor progression, which correlated with the inability to deplete intratumoral Treg. We suggest that in the autochthonous melanoma genesis, other immunosuppressive cells could play an important role and replace immunosuppressive, tumor-promoting functions of Treg. Therefore, effective melanoma immunotherapy should include the inhibition of Treg migration into the tumor combined with neutralization of other immunosuppressive cells and factors in the tumor microenvironment.

Therapeutic Vaccines for Human Papillomaviruses

Although papillomavirus infections are not very immunogenic there is evidence that the immune system controls the spread of virus and the development of diseases associated with such infections. Certain types of human papillomaviruses (HPV) are the major cause of premalignant and malignant diseases of the anogenital tract, most notably cancer of the uterine cervix, a major health care problem worldwide. Since the viral oncoproteins E6 and E7 are constitutively expressed within the tumor cells, they are considered as suitable targets for attack by T lymphocytes. Several approaches to specifically trigger a cell-mediated immune response have been successful in experimental animals, leading to suppression of HPV-induced tumors. First clinical trials have been completed which raise hopes that a similar effect can also be achieved by therapeutic vaccination of humans.

The role of microRNAs in melanoma.

Melanoma is the most dangerous form of skin cancer, being largely resistant to conventional therapies at advanced stages. Understanding the molecular mechanisms behind this disease might be the key for the development of novel therapeutic strategies. MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally control gene expression, thereby regulating various cellular signaling pathways involved in the initiation and progression of different cancer types, including melanoma. In this review, we summarize approaches for the identification of candidate miRNAs and their target genes and review the functions of miRNAs in melanoma. Finally, we highlight the recent progress in pre-clinical use of miRNAs as prognostic markers and therapeutic targets.

T cell response to human papillomavirus 16 E7 in mice: Comparison of Cr release assay, intracellular IFN-γ production, ELISPOT and tetramer staining

Successful vaccination against infections by high-risk papillomaviruses aiming at the prevention of cervical cancer most likely requires the induction of neutralizing antibodies and human papillomavirus (HPV)-specific T cells directed against early viral proteins such as E7. Whereas the technology for detection of antibodies is well established, measurement of T cells is more cumbersome and standardization of assays is difficult. By using chromium release assay, ELISPOT, tetramer staining and intracellular IFN-γ assay, we compared the levels of HPV 16 E7-specific T cells obtained after immunization of C57BL/6 mice with different DNA expression vectors. We found that all four assays gave highly comparable results. ELISPOT can be recommended for future studies as it indicates the presence of activated (i.e. IFN-γ-secreting) T cells in a quantitative manner and combines high sensitivity with relatively low T cell demand.

Screening of human tumor antigens for CD4 T cell epitopes by combination of HLA-transgenic mice, recombinant adenovirus and antigen peptide libraries.

Background As tumor antigen-specific CD4+ T cells can mediate strong therapeutic anti-tumor responses in melanoma patients we set out to establish a comprehensive screening strategy for the identification of tumor-specific CD4+ T cell epitopes suitable for detection, isolation and expansion of tumor-reactive T cells from patients. Methods and Findings To scan the human melanoma differentiation antigens TRP-1 and TRP-2 for HLA-DRB1*0301-restricted CD4+ T cell epitopes we applied the following methodology: Splenocytes of HLA-DRB1*0301-transgenic mice immunized with recombinant adenovirus encoding TRP-1 (Ad5.TRP-1) or TRP-2 (Ad5.TRP-2) were tested for their T cell reactivity against combinatorial TRP-1- and TRP-2-specific peptide libraries. CD4+ T cell epitopes thus identified were validated in the human system by stimulation of peripheral blood mononuclear cells (PBMC) from healthy donors and melanoma patients. Using this strategy we observed that recombinant Ad5 induced strong CD4+ T cell responses against the heterologous tumor antigens. In Ad5.TRP-2-immunized mice CD4+ T cell reactivity was detected against the known HLA-DRB1*0301-restricted TRP-260–74 epitope and against the new epitope TRP-2149–163. Importantly, human T cells specifically recognizing target cells loaded with the TRP-2149–163-containing library peptide or infected with Ad5.TRP-2 were obtained from healthy individuals, and short term in vitro stimulation of PBMC revealed the presence of epitope-reactive CD4+ T cells in melanoma patients. Similarly, immunization of mice with Ad5.TRP-1 induced CD4+ T cell responses against TRP-1-derived peptides that turned out to be recognized also by human T cells, resulting in the identification of TRP-1284–298 as a new HLA-DRB1*0301-restricted CD4+ T cell epitope. Conclusions Our screening approach identified new HLA-DRB1*0301-restricted CD4+ T cell epitopes derived from melanoma antigens. This strategy is generally applicable to target antigens of other tumor entities and to different HLA class II molecules even without prior characterization of their peptide binding motives.