No abstract

Dendritic cells (DC) are professional antigen-presenting cells that play a pivotal role in the induction of immunity. Ex vivo-generated, tumour antigen-loaded mature DC are currently exploited as cancer vaccines in clinical studies. However, antigen loading and maturation of DC directly in vivo would greatly facilitate the application of DC-based vaccines. We formerly showed in murine models that radiofrequency-mediated tumour destruction can provide an antigen source for the in vivo induction of anti-tumour immunity, and we explored the role of DC herein. In this paper we evaluate radiofrequency and cryo ablation for their ability to provide an antigen source for DC and compare this with an ex vivo-loaded DC vaccine. The data obtained with model antigens demonstrate that upon tumour destruction by radiofrequency ablation, up to 7% of the total draining lymph node (LN) DC contained antigen, whereas only few DC from the conventional vaccine reached the LN. Interestingly, following cryo ablation the amount of antigen-loaded DC is almost doubled. Analysis of surface markers revealed that both destruction methods were able to induce DC maturation. Finally, we show that in situ tumour ablation can be efficiently combined with immune modulation by anti-CTLA-4 antibodies or regulatory Tcell depletion. These combination treatments protected mice from the outgrowth of tumour challenges, and led to in vivo enhancement of tumour-specific T-cell numbers, which produced more IFN-g upon activation. Therefore, in situ tumour destruction in combination with immune modulation creates a unique, 'in situ DC-vaccine' that is readily applicable in the clinic without prior knowledge of tumour antigens.

The suppressive activity of regulatory T cells (Treg) has been implicated as an important factor limiting immune mediated destruction of tumor cells. However, not much is known about the presence and function of Treg within tumors. Here we show in a syngeneic murine glioma model a time-dependent accumulation of CD41FoxP31 Treg in brain tumors. Further analysis revealed a time-dependent upregulation of CD25, CTLA-4, GITR and CXCR4 on intratumoral CD41FoxP31 Treg during tumor growth. Moreover, freshly isolated intratumoral Treg were highly suppressive when tested directly ex vivo. Treatment with anti-CD25 monoclonal antibodies (mAbs) significantly reduced the number of these highly suppressive CD41FoxP31 cells within the growing tumor and provoked a CD4 and CD8 T cell dependent destruction of the glioma cells. Combining Treg depletion with administration of blocking CTLA-4 mAbs further boosted gliomaspecific CD41 and CD81 effector T cells as well as antiglioma IgG2a antibody titers resulting in complete tumor eradication without any signs of autoimmunity. These data illustrate that intratumoral accumulation and activation of CD41FoxP31 Treg act as a dominant immune escape mechanism for gliomas and underline the importance of controlling tumor-infiltrating Treg in glioma immunotherapy. ' 2007 Wiley-Liss, Inc. Key words: glioma; regulatory T cell; immune escapeThe naturally occurring CD41 regulatory T cells (Treg) are continuously produced by the thymus and gradually settled in secondary lymphoid organs. They constitute 5-15% of the overall CD41 T cell population and exert a strong suppressive activity on multiple components of the immune system. Although their repertoire in antigen recognition is quite diverse, they preferentially recognize tissue specific self-antigens. As many tumors express self-antigens, CD41 Treg do not only dominantly suppress the activation and expansion of different effector cells capable of mediating autoimmunity, but also effective antitumor responses. 1 So far the most specific marker for naturally occurring CD41 Treg, at least in mice, is FoxP3, a member of the forkhead family of DNA-binding transcription factors. FoxP3 is highly expressed in naturally occurring CD41 Treg and clearly linked to their suppressive function. [2][3][4] Other molecules that are constitutively expressed on CD41 Treg are the IL-2 receptor a-chain (CD25), cytotoxic lymphocyte-associated antigen-4 (CTLA-4) and different members of the tumor necrosis factor (TNF) receptor superfamily like the glucocorticoid-induced TNF related protein (GITR). These molecules, however, do not uniquely distinguish the CD41 Treg from conventional CD41 T cells that can temporally upregulate these molecules following activation. [5][6][7][8][9] Recent data also suggest an important role of Toll-like receptors (TLRs) in controlling Treg. 10 TLR2-triggering on murine Treg in combination with T cell receptor ligation resulted, both in vitro and in vivo, in proliferation of the otherwise anergic Treg. Moreover, the suppressive phenotype o...

Dendritic cells (DC) are professional antigen-presenting cells that play a pivotal role in the induction of immunity. Ex vivogenerated, tumor antigen-loaded mature DC are currently exploited as cancer vaccines in clinical studies. However, antigen loading and maturation of DC directly in vivo would greatly facilitate the application of DC-based vaccines. We have previously shown that in situ tumor destruction by ablative treatments efficiently delivers antigens for the in vivo induction of antitumor immunity. In this article, we show that although 20% of the draining lymph node DCs acquire intratumorally injected model antigens after in situ cryoablation, only partial protection against a subsequent tumor rechallenge is observed. However, we also show that a combination treatment of cryoablation plus TLR9 stimulation via CpG-oligodeoxynucleotides is far more effective in the eradication of local and systemic tumors than either treatment modality alone. Analysis of the underlying mechanism revealed that in situ tumor ablation synergizes with TLR9 stimulation to induce DC maturation and efficient crosspresentation in tumor-bearing mice, leading to superior DC function in vivo. Therefore, in situ tumor destruction in combination with CpG-oligodeoxynucleotide administration creates a unique ''in situ DC vaccine'' that is readily applicable in the clinic. (Cancer Res 2006; 66(14): 7285-92)

Local TLR stimulation is an attractive approach to induce antitumor immunity. In this study, we compared various TLR ligands for their ability to affect murine GL261 cells in vitro and to eradicate established intracerebral murine gliomas in vivo. Our data show that GL261 cells express TLR2, TLR3, and TLR4 and respond to the corresponding TLR ligands with increasing MHC class I expression and inducing IL-6 secretion in vitro, while TLR5, TLR7, and TLR9 are essentially absent. Remarkably, CpG-oligonucleotides (CpG-ODN, TLR9) appeared to inhibit GL261 cell proliferation in a cell-type specific, but CpG-motif and TLR9-independent manner. A single intratumoral injection of CpG-ODN most effectively inhibited glioma growth in vivo and cured 80% of glioma-bearing C57BL/6 mice. Intratumoral injection of Pam3Cys-SK4 (TLR1/2) or R848 (TLR7) also produced a significant survival benefit, whereas poly(I:C) (TLR3) or purified LPS (TLR4) stimulation alone was not effective. Additional studies using TLR9+/+ wild-type and TLR9−/− knockout mice revealed that the efficacy of local CpG-ODN treatment in vivo required TLR9 expression on nontumor cells. Additional experiments demonstrated increased frequencies of tumor-infiltrating IFN-γ producing CD4+ and CD8+ effector T cells and a marked increase in the ratio of CD4+ effector T cells to CD4+FoxP3+ regulatory T cells upon CpG-ODN treatment. Surviving CpG-ODN treated mice were also protected from a subsequent tumor challenge without further addition of CpG-ODN. In summary, this study underlines the potency of local TLR treatment in antiglioma therapy and demonstrates that local CpG-ODN treatment most effectively restores antitumor immunity in a therapeutic murine glioma model.

Both melanoma and glioma cells are of neuroectodermal origin and share common tumor associated antigens. In this article, we report that the melanocyte differentiation antigen TRP2 (tyrosinase-related protein 2) is not predominately involved in the tumor rejection of a syngeneic murine glioma. Although GL261 glioma cells endogenously expressed TRP2 and were lysed by TRP2 specific cytotoxic T cells (CTLs) in vitro, vaccinations with TRP2 peptide-pulsed dendritic cells (DCs) could only induce minor antiglioma responses in a prophylactic setting and failed to work in a stringent setting where vaccine and tumor were administered on the same day. Further analysis revealed that TRP2 is not recognized by bulk CTLs after depletion of regulatory T cells which results in tumor rejections in vivo. In contrast to TRP2 peptidepulsed DC, tumor lysate-pulsed DCs were more potent as a vaccine and completely protected mice from tumor outgrowth in a prophylactic setting. However, the vaccine efficacy of tumor lysate-pulsed DC was not sufficient to prevent the tumor outgrowth when tumors were inoculated the same day. In this case, Treg depletion before vaccination was essential to boost antiglioma immune responses leading to the rejection of 80% of the mice and long-term immunity. Therefore, we conclude that counteracting the immunosuppressive glioma tumor environment via depletion of regulatory T cells is a prerequisite for successful eradication of gliomas after targeting multiple tumor antigens by using tumor lysate-pulsed DCs as a vaccine in a more stringent setting. ' 2007 Wiley-Liss, Inc.Key words: glioma; TRP2; tumor lysate; dendritic cell; regulatory T cell; CD25-depletion Dendritic cell (DC)-based immunotherapy is regarded as an attractive strategy for the therapy of malignant gliomas. Several animal studies have demonstrated that DCs can efficiently mediate the induction of antitumor responses within the central nervous system. 1-9 A number of phase I/II studies have been performed and others are ongoing to evaluate and improve DC vaccines for patients with malignant gliomas. 10-15Although DC-based immunotherapies for various cancers including malignant gliomas led to the induction of antitumor cytotoxic T cells (CTLs) and lymphocyte infiltration into tumors in situ, objective tumor regressions have been limited to a few cases. 16 In addition, most mouse glioma models primarily assessed the ability of DC-based immunotherapy to prevent the outgrowth of tumor in a prophylactic setting in which tumor cells are injected after vaccination. Attempts to treat tumors at more stringent conditions, like administration of tumor cells and vaccine on the same day (simultaneous setting) or a therapeutic setting were either not evaluated or shown to be unsuccessful, despite the presence of large numbers of antiglioma immune cells.A major obstacle for an active immunization against solid tumors is the presence of immunosuppressive factors at the local tumor environment. There is accumulating evidence that active suppressor mechanisms ar...

Immunostimulatory cytidyl guanosyl (CpG) motifs are of great interest as cancer vaccine adjuvants. They act as potent inducers of Th1 responses, including the activation of cytotoxic CD8 + T lymphocytes (CTL). Whereas animal models have provided clear evidence that CpG enhances antitumor immunity, clinical trials in humans have thus far been less successful. Applying cryosurgery as an instant in situ tumor destruction technique, we now show that timing of CpG administration crucially affects colocalization of antigen and CpG within EEA-1 + and LAMP-1 + compartments within dendritic cells in vivo. Moreover, antigen/CpG colocalization is directly correlated with antigen cross-presentation, the presence of CTL, and protective antitumor immunity. Thus, failure or success of CpG as a vaccine adjuvant may depend on colocalization of antigen/CpG inside DCs and hence on the timing of CpG administration. These data might aid in the design of future immunotherapeutic strategies for cancer patients. [Cancer Res 2008;68(13):5390-6]

The mammalian small heat shock protein aB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wildtype, nonphosphorylatable and all possible pseudophosphorylation mutants of aB-crystallin by immunoblot and immunocytochemical analyses of stable and transiently transfected cells. We observed that pseudophosphorylation at two (especially S19D/S45D) or all three (S19D/S45D/ S59D) sites induced the partial translocation of aB-crystallin from the detergent-soluble to the detergent-insoluble fraction. Double immunofluorescence studies showed that the pseudophosphorylation mutants localized in nuclear speckles containing the splicing factor SC35. The aB-crystallin mutants in these speckles were resistant to mild detergent treatment, and also to DNase I or RNase A digestion, indicating a stable interaction with one or more speckle proteins, not dependent on intact DNA or RNA. We further found that FBX4, an adaptor protein of the ubiquitin-protein isopeptide ligase SKP1/CUL1/F-box known to interact with pseudophosphorylated aB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody against aB-crystallin endogenously phosphorylated at Ser45, our findings suggest that aB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles.