Erik Hooijberg

Glycan-modified liposomes boost CD4 + and CD8 + T-cell responses by targeting DC-SIGN on dendritic cells

Cancer immunotherapy requires potent tumor-specific CD8(+) and CD4(+) T-cell responses, initiated by dendritic cells (DCs). Tumor antigens can be specifically targeted to DCs in vivo by exploiting their expression of C-type lectin receptors (CLR), which bind carbohydrate structures on antigens, resulting in internalization and antigen presentation to T-cells. We explored the potential of glycan-modified liposomes to target antigens to DCs to boost murine and human T-cell responses. Since DC-SIGN is a CLR expressed on DCs, liposomes were modified with DC-SIGN-binding glycans Lewis (Le)(B) or Le(X). Glycan modification of liposomes resulted in increased binding and internalization by BMDCs expressing human DC-SIGN. In the presence of LPS, this led to 100-fold more efficient presentation of the encapsulated antigens to CD4(+) and CD8(+) T-cells compared to unmodified liposomes or soluble antigen. Similarly, incubation of human moDC with melanoma antigen MART-1-encapsulated liposomes coated with Le(X) in the presence of LPS led to enhanced antigen-presentation to MART-1-specific CD8(+) T-cell clones. Moreover, this formulation drove primary CD8(+) T-cells to differentiate into high numbers of tetramer-specific, IFN-γ-producing effector T-cells. Together, our data demonstrate the potency of a glycoliposome-based vaccine targeting DC-SIGN for CD4(+) and CD8(+) effector T-cell activation. This approach may offer improved options for treatment of cancer patients and opens the way to in situ DC-targeted vaccination.

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.

Extent and location of tumor infiltrating lymphocytes in microsatellite stable colon cancer predict outcome to adjuvant active specific immunotherapy

Meeting abstractsnnTo determine the prognostic and predictive value of tumor infiltrating lymphocytes (TIL) in colon cancer in a cohort of patients who previously took part in a trial on adjuvant Active Specific Immunotherapy (ASI).nnVermorken et al.[[1][1]] conducted a multicenter clinical trial on

Abstract 757: Transfection of dendritic cells with IL-21 mRNA gives rise to CD8+ effector T cells with a higher proliferation rate and increased cytotoxic capacity

Interleukin -21, mainly produced by activated CD4+ T cells, has been shown to play a central role in the differentiation and proliferation of B and T cells. Here, we investigated the utility of IL-21 to generate high numbers of functional CD8+ effector T cells for the immunotherapy of cancer. To this end, we made use of IL-21 mRNA transfected dendritic cells (DCs) to prime and activate CD8+ T cells recognizing the melanoma antigen Mart-1. Expansion rates and functional avidity were assessed. mRNA was transcribed in vitro from pGEM encoding eGFP/2A/IL-21 or IL-21. IL-21 transfection of cytokine-matured DCs was performed, which did not alter the expression of co-stimulatory markers (CD40, CD80, CD83 and CD86). Translation of the mRNA and secretion of the IL-21 protein were confirmed by ELISA. Initially, CD28+ T cell bulks were stimulated with allogeneic DCs transfected with IL-21 or eGFP (i.e. negative control) mRNA. The T cells primed in the presence of IL-21 showed a higher proliferation rate and cytotoxic capacity (by Granzyme B expression) than controls. Subsequently, IL-21 mRNA was co-transfected with mRNA encoding a MART-1 minigene, consisting of four copies of the MART-126L-35 immunodominant epitope preceded by a Ubiquitin sequence, into mature DCs. These DCs were used for the priming of isolated autologous CD8β+ T cells. After two rounds of stimulation, a slight, but not significant, increase in the frequencies of HLA-A2-tetramer binding T cells was observed for the MART-126L-35 epitope in comparison to control cultures with MoDCs transfected with the Ubi-MART-1 minigene mRNA without IL-21 mRNA. Of note, these specifically primed tetramer+ T cells displayed an equivalent functional avidity by IFNγ release as compared to their counterparts in control cultures, but did express significantly higher levels of Granzyme-B (P=0.05), indicative of a higher cytotoxic capacity. To assess the effects of IL-21 stimulation on effector-memory T cells, we made use of an established MART-126L-35 specific T cell clone. In multiple parallel cultures, significantly increased proliferation rates and Granzyme-B expression levels were observed when the T cells were stimulated by MoDCs co-transfected with both Ubi-MART-1 and IL-21 mRNA, as compared to Ubi-MART-1 mRNA only. We conclude that the inclusion of IL-21 in active vaccination approaches, such as mRNA-based DC vaccines, or indeed in adoptive T cell therapies, may increase their efficacy through the accelerated expansion of anti-tumor CD8+ effector T cells with increased cytolytic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 757. doi:10.1158/1538-7445.AM2011-757