The identification and characterisation of tumour-associated antigens and the rapid growth in knowledge of immune system function, has laid the foundations for developing effective protocols for the immune therapy of cancers. Immunotherapy has the potential to eradicate systemic tumour in multiple sites through the body and the specificity to discriminate between neoplastic and non-neoplastic cells. Adoptive immunotherapy strategies have been established whereby cells are stimulated to recognise tumour antigens (Ags) ex vivo and reinfused into patients. Spectacular results have been achieved in some individuals using this form of therapy; however, response rates and durations have been limited. To develop better immunotherapeutic protocols, more information is required on the interactions that occur between the immune system and the developing cancer. Studies on the cells responsible for tumour regression following adoptive transfer are limited and the mechanisms by which rejection occurs is not well characterised.
This study investigates aspects of adoptive immunotherapy that will best predict successful regression of an established transplantable tumour. Human papillomavirus- 16 (HPV16) infection has been associated with the development of anogenital cancers and requires the continued expression of the E7 oncoprotein. This protein can therefore act as a non-self target Ag for recognition by the host immune system. It was an aim of these studies to investigate aspects of adoptive immunotherapy, with a particular emphasis on comparing and contrasting the efficacy of Ag-specific CTL when transferred in a mixed, multicellular preparation compared to long-term cultured CTL. Hypotheses to be tested include:
1) Tumour-antigen-specific adoptive immunotherapy is necessary for the inhibition of established tumour growth.
2) The effector cell type, and mechanism of tumour growth inhibition, varies depending on the stage of tumour development.
3) Ex vivo lymph node cell preparations are more effective at limiting tumour growth than long-term cultured CTL.
The experimental model system involves the transfer of cells from the draining lymph nodes of immunised mice or from cultured E7-specific CTL lines, into mice bearing an established E7-expressing tumour. Success of therapy was assessed by measurement of explanted tumour weights after set time periods. It was shown that the administration of lymph node cells (LNCs) via the intravenous route gave the best tumour inhibitory effect when transferred at day 4; however, this route proved ineffective at reducing tumours that had been established for only 1 day, suggesting that tumour vascularisation is required for effective adoptive therapy by intravenous transfer. LNCs from mice immunised with the E7-tumour Ag or with non-tumour Ag proteins in adjuvant were both able to significantly reduce the resultant tumour mass when transferred into mice with 4-day tumours. In contrast, at 8 days after tumour challenge only the transfer of tumour-Ag-specific LNCs had any therapeutic effect. Compared with non-specific therapy, specific cellular therapy resulted in slightly longer lasting effects, and was more successful at retarding the growth of more established tumours. Therefore, there exists a window of opportunity where non-tumour specific lymphocyte transfers can be therapeutic, although this effect is limited.
Therapy of both 4-day and 8-day established tumours was found to be CD8-dependent and CD4-independent. When CD8⁺ cells were depleted from the LNC populations and transferred into SCID mice with an 8-day established tumour, the protective effect was not completed inhibited but when transferred into Rag knockout mice, depletion of CD8⁺ cells completed abrogated tumour growth inhibition. However, when both CD4⁺ and CD8⁺ cells were depleted, the protective effect of LNC transfers was restored. This suggests that there exists a CD4⁺ suppressive population of cells that regulates the function of effector cells or that additional mechanism of tumour growth inhibition are operating at this later stage of tumour growth. Given the diversity in therapeutic efficacy dependent on differences in the specificity of the transferred LNC preparations, the mechanism of tumour growth inhibition was examined using donor mice in which key effector molecules had been deleted. It was found that the mechanism of tumour growth inhibition by donor LNCs from E7-immunised mice at day 8 did not require the production of either perforin or γ-interferon, but did to some extent utilise the Fas/FasL pathway. It was essential that non-specifically immunised donor mice were able to produce both perforin and γ -interferon to generate a response, when LNCs were transferred at day 4, whereas tumour-specific LNCs required an ability to produce perforin for effector function. LNCs from Jα28r‐/‐ chain knockout (NKT cell KO) mice did not convey any ability to reduce tumour burden after a day 4 transfer but were effective when transferred into mice with an 8-day established tumour. Therefore, for a reduction in tumour mass to occur, there appears to be a requirement for the presence of NKT cells in the donor cellular preparation. However, this requirment was only seen at particular stages of tumour growth. Consequently, the efficacy and mechanism of tumour growth inhibition was found to be dependent on the timing of adoptive transfer, in relation to tumour development, and the specificity of the transferred cells.
Results comparing LNC preparations and cultured CTL suggested that not all cultured CTL lines were as effective at reducing tumour mass following adoptive transfer, particularly when the murine hosts were immunocompromised. Studies have emphasised the diverse nature of an anti-tumour immune reaction, which involves a finely tuned interactive response and depends on multiple regulatory and effector cell types. Consequently, the challenge now for tumour immunologists is the development of immunotherapeutic strategies against cancer that incorporate the multifaceted nature of anti-tumour responses.