Research paperBlocking IL-10 signalling at the time of immunization renders the tumour more accessible to T cell infiltration in mice
Introduction
Human papillomavirus (HPV) infection accounts for approximately 5.3% of cancers worldwide, including cervical cancer and some of the genital, head and neck cancers [1], [2], [3], [4]. It is estimated that 50 million women carry HPV and approximately 500,000 women develop cancer each year [5]. HPV type 16 and 18 infection is associated with 70% of cervical cancer. The prophylactic vaccine against HPV infection (Gardasil®, Cervarix®) was introduced to the market in 2006 and is effective at preventing persistent HPV16/18 infection [6], [7].
However, Gardasil® and Cervarix® are ineffective for women who had already been infected with HPV [1]. Existing treatments for cervical cancer, such as surgery, chemotherapy and radiotherapy often fail to prevent recurrent diseases and sometimes result in significant side effects. As a consequence, there is an urgent need to develop better strategies to treat chronic HPV infection related tumours [8], [9], [10].
The ideal cancer therapeutic vaccine, which would cure people who already have cancer, is in an active state of investigation [11], [12], [13]. Compared with surgery, radio- or chemo-therapy treatment, cancer therapeutic vaccine targets tumour cells without harming normal cells, tissues or organs [14], [15]. However, vaccines often fail to promote the regression of tumours in clinical trials [10], [15], [16], [17], [18]. The low therapeutic efficacy of cancer vaccines may be due to tumour-induced immune escape mechanisms and the immune suppressive tumour microenvironments [10], [19], [20]. Tumour cells often express high levels of immune suppressive molecules, such as PD-L1 [21], [22], [23], and reduced MHC molecules on their cell membranes [24], [25]. Immune suppressive myeloid-derived suppressor cells, tumour infiltrating regulatory T cells, tumour associated macrophage and immune suppressive cytokines, such as interleukin 10 and TGFβ, can inhibit cytotoxic T cells from killing tumour cells [26], [27], [28], [29], [30]. Therefore, an effective response that is able to lead tumour regression due to a therapeutic vaccine, requires inducing successful specific cytotoxic T lymphocytes (CTLs). These effector T cells are able to migrate to tumour sites, overcome the hostile tumour microenvironment and kill the tumour cells [31], [32].
Recently, therapeutic vaccines against chronic human papillomavirus infection related pre-cancer have shown efficacy [33], [34], [35]. Synthetic long E6/E7 peptide formulated in incomplete Freund’s adjuvant (IFA), chimeric papillomavirus capsid protein L1L2E7 and E7 DNA vaccines, resulted in pre-cancer cervical intraepithelial neoplasia (CIN) and vulvar intraepithelial neoplasia (VIN) regression in clinical trials [33], [34], [35], [36], [37]. Of the three trials that show clinical efficacy only the long E6/E7 peptide/IFA trial is randomized, double blind and placebo controlled. L1L2E7 vaccine requires simultaneously local administration of the immune response modifier imiquimod which is a Toll like receptor 7 ligand, to be effective [36]. Vaccine induced CD8+ T cell responses, are critical for the regression of CIN or VIN [20], [33], [34], [37].
Interleukin 10 is a cytokine with the ability to control or terminate immune responses to both self and foreign antigens [38]. Temporal IL-10 blockade at the time of immunization enhances nucleotide, peptide, papillomavirus-like particles induced CD8+ T cell responses [39], [40], [41], [42], [43] and clears chronic viral infection more efficiently than without IL10 signalling blockade [44], [45], [46]. Recently, it was demonstrated that a candidate adenovirus vectored-HIV-1 vaccine elicits stronger CD8+ T cell responses to its delivered HIV conserved regions when IL-10 signalling is temporal blocked [47]. Moreover, MPL, a detoxified from of LPS, has similar ability to induce CD8+ T cell responses as LPS when vaccination and blocking IL-10 signalling simultaneously [41]. Administration of anti-IL-10R antibodies through subcutaneously injection also enhances vaccine induced CD8+ T cell responses [41]. All these results suggest that this immunization strategy may be effective against human chronic viral infection and chronic viral infection related cancers and warrant further investigation.
We demonstrated recently [41] that immunization with long E7 peptide/LPS and temporal blockade of IL-10 signalling with anti-IL-10R antibodies prevents HPV16 E7 transformed TC-1 tumour growth in both prophylactic and therapeutic settings in mice, similar to that resulted from immunization with synthetic long E7 peptide/IFA. In this paper, we investigated the memory CD8+ T cell responses elicited by immunization and blocking IL10 signalling simultaneously, and compared vaccine induced CD8+ T cell responses, T cell infiltration to tumour site after either long E7 peptide/IFA or long E7 peptide/LPS/anti-IL10R immunization.
Section snippets
Mice
Six to eight-week old adult female C57BL/6 (H-2b) mice were purchased specific pathogen free (SPF) and kept under SPF conditions throughout at the Animal Resource Centre, Sun Yat-Sen University, Guangdong province, China. The mice were fed irradiated food and autoclaved water and kept in 12 h light and dark cycles. All experiments were approved by and performed in compliance with the guidelines of Foshan First Peoples’ Hospital Animal Experimentation Ethics Committee. Mice were 3–5 mice per
Long HPV16 E7 peptide/LPS immunization and simultaneous IL-10 signalling blockade elicits stronger antigen specific IFNγ+CD8+ T cell responses
IFA has been in the principal choice of adjuvant in many peptide-based vaccines [50], [51]. To compare T cell responses induced by blockade of IL10 signalling at the time of immunization with IFA-based immunization, C57BL/6 mice were primed and boosted with long E7 peptide/IFA or long E7 peptide/LPS/aIL10R subcutaneously twice on day 0 and day 7. Antigen specific IFNγ+CD8+ T cell responses were determined by ELISPOT assay and the numbers of splenic IFNγ+CD8+ T cell and IFNγ+CD4+ T cells were
Discussion
In this paper, we compared vaccine induced cytotoxic T cell responses after immunization with either long E7 peptide/LPS/aIL-10R antibody or long E7 peptide/IFA. Long E7 peptide/LPS/aIL-10R immunization elicited higher numbers of antigen specific CD8+ T cell responses than did E7/IFA. This immunization strategy also leads to more IFNγ secreting CD4+ and CD8+ T cells trafficking to tumour sites. Moreover, priming with long E7 peptide/LPS/aIL-10R, and then boosting with long E7 peptide/IFA,
Acknowledgments
The authors wish to thank Prof. Richard Burns for critical reading of the manuscript. The current research was supported in part by Science and Technology Research program of Foshan city (No.: 2012B03180003); Foshan City Council Research Platform project (No: 26), Science and technology research program of Guangdong province (No.: 2012AA100461) to XSL and National Natural Science Foundation of China (No.: 81472451) to XSL and TFW.
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