Annelies W. Turksma

Effector memory T-cell frequencies in relation to tumour stage, location and HPV status in HNSCC patients

BACKGROUND The immune system plays an important role in tumour immune surveillance. Head and neck squamous cell carcinoma patients are often immune compromised. OBJECTIVE To chart the baseline levels of T-cell subpopulation frequencies in patients with cancer prior to treatment. SUBJECTS AND METHODS Blood samples of patients were taken at the time of diagnosis, analysed with flowcytometry and compared with blood samples of healthy donors. RESULTS Compared to healthy donors, a significant shift from naive to effector memory T cells was observed. This effect was most prominent in stage II patients. A similar shift from naive to effector memory T cells was noted in patients with oropharynx or larynx squamous cell carcinomas. Furthermore, the percentage of effector memory and effector T cells was higher in the group of patients with human papillomavirus-positive oropharyngeal squamous cell carcinomas, compared with patients with human papillomavirus-negative tumours, suggestive of virus-induced T-cell activation. CONCLUSION Here, we provide a simple and easily implementable tool to document T lymphocyte subsets in the peripheral blood of head and neck cancer patients, which might be useful for prognosis and/or therapy response prediction.

Generating HPV specific T helper cells for the treatment of HPV induced malignancies using TCR gene transfer.

BackgroundInfection with high risk Human Papilloma Virus (HPV) is associated with cancer of the cervix, vagina, penis, vulva, anus and some cases of head and neck carcinomas. The HPV derived oncoproteins E6 and E7 are constitutively expressed in tumor cells and therefore potential targets for T cell mediated adoptive immunotherapy. Effective immunotherapy is dependent on the presence of both CD4+ and CD8+ T cells. However, low precursor frequencies of HPV16 specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer purposes. An alternative to generate HPV specific CD4+ and CD8+ T cells is TCR gene transfer.MethodsHPV specific CD4+ T cells were generated using either a MHC class I or MHC class II restricted TCR (from clones A9 and 24.101 respectively) directed against HPV16 antigens. Functional analysis was performed by interferon-γ secretion, proliferation and cytokine production assays.ResultsIntroduction of HPV16 specific TCRs into blood derived CD4+ recipient T cells resulted in recognition of the relevant HPV16 epitope as determined by IFN-γ secretion. Importantly, we also show recognition of the endogenously processed and HLA-DP1 presented HPV16E6 epitope by 24.101 TCR transgenic CD4+ T cells and recognition of the HLA-A2 presented HPV16E7 epitope by A9 TCR transgenic CD4+ T cells.ConclusionOur data indicate that TCR transfer is feasible as an alternative strategy to generate human HPV16 specific CD4+ T helper cells for the treatment of patients suffering from cervical cancer and other HPV16 induced malignancies.

IL-21 in cancer immunotherapy: At the right place at the right time

Interleukin-21 (IL-21) has been described as a potent stimulator of antitumor T-cell immunity, but also of autoimmune reactions and oncogenesis. Antigen presenting cells genetically modified to release IL-21 allow for the expansion of tumor-specific T cells exhibiting favorable effector and growth characteristics and a minimal risk of detrimental side effects.

Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients.

BackgroundNew treatment modalities are needed for the treatment of cancers of the head and neck region (HNSCC). Survivin is important for the survival and proliferation of tumor cells and may therefore provide a target for immunotherapy. Here we focused on the ex vivo presence and in vitro induction of survivin specific T cells.MethodsTetramer staining and ELIspot assays were used to document the presence of survivin specific T cells in patient derived material, and to monitor the presence and persistence of survivin specific T cells after repeated in vitro stimulation with autologous dendritic cells.ResultsEx vivo analysis showed the presence of survivin-specific T cells in the peripheral blood (by tetramer analysis) and in the draining lymph node (by ELIspot analysis) in a HNSCC and a locally advanced breast cancer patient respectively. However, we were unable to maintain isolated survivin specific T cells for prolonged periods of time. For the in vitro generation of survivin specific T cells, monocyte derived DC were electroporated with mRNA encoding full length survivin or a survivin mini-gene together with either IL21 or IL12 mRNA. Western blotting and immunohistochemical staining of dendritic cell cytospin preparations confirmed translation of the full length survivin protein. After repeated stimulation we observed an increase, followed by a decrease, of the number of survivin specific T cells. FACS sorted or limiting dilution cloned survivin specific T cells could not be maintained on feeder mix for prolonged periods of time. Protein expression analysis subsequently showed that activated, but not resting T cells contain survivin protein.ConclusionsHere we have shown that survivin specific T cells can be detected ex vivo in patient derived material. Furthermore, survivin specific T cells can be induced in vitro using autologous dendritic cells with enforced expression of survivin and cytokines. However, we were unable to maintain enriched or cloned survivin specific T cells for prolonged periods of time. Endogenous expression of survivin in activated T cells and subsequent fratricide killing might explain our in vitro observations. We therefore conclude that survivin, although it is a universal tumor antigen, might not be the ideal target for immunotherapeutic strategies for the treatment of cancer of the head and neck.

Increased cytotoxic capacity of tumor antigen specific human T cells after in vitro stimulation with IL21 producing dendritic cells.

Monocyte derived dendritic cells (moDC) electroporated with tumor associated antigen derived mRNA can elicit specific T cells against tumor cells in vivo. IL21 has been shown to enhance activation and cytotoxicity in CD8+ T cells. We therefore investigated in vitro effects on human CD8+ T-cells after stimulation with IL21 mRNA electroporated moDC. Codon modification of the IL21 gene significantly enhanced IL21 production upon electroporation of moDC. Tumor associated antigen specific CTL induction efficiency was significantly enhanced when codon modified IL21 mRNA was co-electroporated with tumor associated antigen mRNA. Tumor associated antigen specific T cells induced by codon modified IL21-DC demonstrated increased cytotoxic capacity and killing compared to control cultures. In conclusion, ectopic expression of codon modified IL21 by moDC enhances the priming efficiency of the DC as well as the cytotoxic potential of the induced CTL.

Abstract 1649: Extent and location of tumor infiltrating lymphocytes in colon cancer predicts outcome to adjuvant active specific immunotherapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: We investigated the prognostic and predictive value of tumor infiltrating lymphocytes (TIL) in colorectal cancer in a cohort of patients who previously took part in a trial on adjuvant Active Specific Immunotherapy (ASI). Experimental Design: We determined the numbers of CD3+ and CD8+ cells in archival tumor samples of 110 colorectal cancer patients as total infiltrating TIL, intraepithelial TIL and stromal TIL. Survival curves were used to determine the prognostic and predictive value of TIL. Results: Patients with microsatellite instable (MSI) tumors had higher numbers of intraepithelial CD3+ and CD8+ TIL compared to patients with microsatellite stable (MSS) tumors. Patients with MSI tumors had a good prognosis. Patients with MSS cancers with high intraepithelial or stromal TIL numbers had a better prognosis compared to those with low TIL numbers. Significant survival benefits were found for the disease specific survival (DSS) and recurrence free interval (RFI) for patients with high infiltrates of stromal CD3+ or intraepithelial CD8+ cells. Additionally high numbers of total CD8+ and high stromal CD8+ cells gave a significant difference in RFI compared to low TIL numbers. The numbers of TIL found in MSS tumors also have predictive value with respect to response to adjuvant ASI treatment. Compared to controls significance was reached in ASI treated patients with high total CD3, high stromal CD3 or high stromal CD8 numbers in DSS as well as RFI. Conclusion: ASI therapy is effective in patients with MSS colorectal tumors harboring high numbers of preexisting stromal CD3 or CD8 positive TIL. Citation Format: Erik Hooijberg, Annelies W. Turksma, Marc Shamier, Kevin Lam, Veerle MH Coupe, Vincent A. de Weger, Jeroen AM Belien, Alfons J. van den Eertwegh, Gerrit A. Meijer, Chris JLM Meijer. Extent and location of tumor infiltrating lymphocytes in colon cancer predicts outcome to adjuvant active specific immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1649. doi:10.1158/1538-7445.AM2014-1649

56INIMMUNOTHERAPY FOR HEAD AND NECK CANCER

ABSTRACT Head and neck squamous cell carcinoma is the sixth most common cancer in the western world. Little improvement in treatment has been made over the last few decades to increase the relatively low 5-year survival rate. This calls for new treatment modalities. First, we charted the baseline levels of T cell subpopulation frequencies in these patients prior to treatment. Compared to healthy donors, a significant shift from naive to effector memory T cells was observed. A similar shift was noted in patients with oropharynx or larynx squamous cell carcinomas. The percentage of effector (memory) T cells was higher in the group of patients with HPV-positive carcinomas, suggestive of virus-induced T-cell activation. Second, HPV derived oncoproteins E6 and E7 are constitutively expressed in tumor cells and therefore are potential targets for T cell mediated adoptive immunotherapy. However, low precursor frequencies of HPV16 specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer purposes. Administration of T cell receptor transgenic T cells may form an alternative. We have isolated HPV specific TCRs and show reactivity against appropriate target cells of TCR transgenic cytotoxic and helper T cells. Third, active immunotherapy consisting of vaccination with mature dendritic cells expressing relevant tumor antigens may be explored. We have shown that survivin specific T cells can be detected ex vivo in patient derived material. Furthermore, survivin specific T cells can be induced in vitro using autologous dendritic cells with enforced expression of survivin and cytokines. However, due to fratricide killing we were unable to maintain enriched or cloned survivin specific T cells for prolonged periods of time in culture. We therefore conclude that survivin, might not be the ideal target. We will discuss options of both passive and active immunotherapy for the treatment of head and neck cancer patients and hurdles yet to overcome. Disclosure: All authors have declared no conflicts of interest.