In this study, we validated in vivo the concept of tumor immunotherapy with ex vivo total RNA-loaded DCs in the context of malignant glioma. Moreover, the inevitable link between active immunotherapy and counteracting immune suppression was also investigated.
This immunotherapeutic approach was implemented in the well-established murine GL261 glioma model. We opted for prophylactic treatment since others reported the very aggressive nature of the GL261 model, necessitating additional intervention in curative settings.44,45
Moreover, this model reflects more the clinical therapeutic setting in which DC vaccination is also given at a stage of minimal residual disease after (sub)total resection and not at the time of bulky tumor disease.14–16
Vaccination with DCm-GL261-RNA prolonged survival and protected nearly half of the treated animals against subsequent tumor challenge. Mapping of weight loss and tumor-induced neurological deficit clearly underscored our survival data.21
Finally, histological analysis revealed DCm-GL261-RNA vaccination resulted in infiltration of lymphocytes and nonlymphoid cells, especially at the interface of the tumor mass with the normal brain parenchyma.
The specificity of any induced immune response represents a major issue when dealing with cellular cancer immunotherapy. The specificity of the immune response induced by RNA-loaded DCs in this model was demonstrated in vivo by challenging DCm-GL261-RNA–treated mice with an immunogenic fibrosarcoma cell line that is embryologically unrelated to glial tumors, or challenging DCm-MC17-51-RNA–treated mice with GL261 tumor cells. In both cases, the DC-mediated immunity against the target tumor resulted in a delay of growth of the target tumor, in contrast to the unchanged growth rate of the nontargeted tumor. Specificity was also confirmed at the level of ex vivo cytotoxic activity of in vivo stimulated brain-infiltrating lymphocytes, which clearly depicted tumor-suppressive activity against the GL261 tumor cells but not against the LLC cell line. However, we noticed that alongside immunization, treatment with DCm-LLC-RNA and DCm-splenocyte-RNA resulted in a prolonged median survival compared to untreated mice. A similar finding was observed in the group of mice treated with mock-loaded DCm. We postulate that the induction of a minor immune response upon tumor challenge itself might be boosted in a nonspecific manner by administration of activated DCs. In none of these control conditions, however, did tumor-challenged mice survive, pointing to the specific immunological protective effect of DCm-GL261-RNA treatment.
The boosted status of the immune system of DCm-GL261-RNA–vaccinated animals was shown by specific ex vivo restimulation of splenocytes and dLN cells. Splenocytes and dLN cells responding to GL261 Ags were retrieved in DCm-GL261-RNA–treated animals and only to a significantly lower extent in untreated mice.
Only half of the treated mice could be protected. With the help of online monitoring of tumor load through in vivo BLI, we recently defined two different subcategories within mice that were not protected by DCm-GL261-RNA treatment.21
One group of animals displayed in vivo flux values similar to those of untreated mice and are hence considered genuine nonresponding mice. On the other hand, we observed a fraction of partially responding mice that exhibited an initial tendency toward tumor rejection but later showed progressive disease. To our view, this is suggestive of a delicate balance between immunogenic antitumor and counteracting tolerogenic and/or suppressive mechanisms involved in the antiglioma immune response. This delicate balance, moreover, might be prone to interanimal variability. We therefore tried to unravel the balance between endogenous and/or vaccine-induced antitumor mechanisms and endogenous and/or tumor-induced tolerogenic or suppressive processes. This was accomplished by in vivo depletion of both CD8+
effector T-cells and CD25+
Treg with aCD8 and aCD25 mAb, respectively.
In accordance with recently published data by Grauer et al.,37
we have shown that CD8+
T-cells are involved both in an endogenous immune response upon tumor challenge and a vaccine-mediated protective antitumor immune response. When CD8+
T-cells were depleted at the time of tumor challenge, immunological protection was completely abolished, and all animals died. However, the median survival of DCm-GL261-RNA–treated CD8-depleted mice was still significantly longer than that of the untreated CD8-depleted mice. This indicates that DC immunotherapy is not solely acting through CD8+
T-cells. Other key players such as CD4+
helper T-cells, natural killer (NK) cells, and NK/T-cells might be likewise affected by this kind of DC treatment.46–48
In our hands, a minor fraction of the ex vivo differentiated DCs from bone marrow progenitor cells that were used for immunization showed low expression of CD8a. Hence, this CD8a+
DC subpopulation is prone to in vivo elimination by the injection of aCD8a mAb 1 week after the second vaccination. The functional consequences of this phenomenon are not clear. The CD8aa homodimer has been regarded as a cell lineage marker rather than a molecule that could contribute to functional differences between (CD8a+
) lymphoid and (CD8a−
) myeloid DC.49
However, Hong et al.50
recently reported that expression of CD8a on bone-marrow–derived DCs may play a functional role in enhancement of T-cell activation.
We noted that a single injection of a CD25-depleting mAb creates a temporal decrease in CD4+
Treg activity that is sufficient to completely protect animals from subsequent (early) IC tumor challenge. This attributes a dominant role to Treg in our experimental glioma model. A direct effect of aCD25 on GL261 cells was excluded, which agrees with recent findings of Curtin et al.,34
who also demonstrated that the efficiency of Treg depletion in a curative setting is dependent on the tumor burden since systemic administration of PC61 has a beneficial effect on survival if applied 15 days after tumor challenge but not when given 24 days after tumor challenge. Other investigators have found that curative DC-based immunotherapy in the GL261 model is efficient only if CD25-expressing Tregs are first depleted.39,51
The crucial role of Treg in glioma and other types of cancer and the impact on immunotherapy has been documented extensively both in humans and in experimental rodent models by many groups.52–55
For an excellent review on this subject, we refer to the work of W. Zou.56
Recent evidence has arisen that aCD25 treatment with the PC61 mAb does not result in a genuine depletion of Tregs but rather a functional inactivation of naive CD69lo
Tregs with rapid internalization and shedding of the IL-2 receptor α unit.57
Considerable caution should be taken into account when CD25 expression on CD4+
T-cells is used to monitor Treg kinetics. Hence, in this study, only FoxP3+
cells were considered the true Treg population. In our experiments, restoration of CD25 expression in CD4+
splenocytes by vaccination after initial CD25 depletion was concomitant with an increase in FoxP3 expression. This suggests that DC treatment is capable of inducing Tregs besides its beneficial influence on the effector arm of cellular immunity. It remains an open question whether these FoxP3-expressing cells are true functional Tregs with in vivo suppressive capacity, since all animals that received combined treatment were long-term survivors from primary tumor challenge.
From rechallenge experiments, we concluded that treatment with DCm-GL261-RNA was able to induce immunological memory against the tumor, since animals that initially received combined treatment (consisting of CD25 depletion and DCm-GL261-RNA vaccination) or survived after treatment with DCm-GL261-RNA alone displayed prolonged survival and were again partially protected against tumor challenge. To our view, the observed early and also late immunological protection against glioma challenge clearly demonstrates the efficiency of immunization with DCs. On the other hand, mice that received aCD25 treatment only were not protected upon rechallenge, so either the vaccine-induced FoxP3-expressing CD4+
cells are nonfunctional Tregs, which is unlikely, or the balance between immunogenicity and tolerance is again sufficiently tilted toward the former at a later time point (day 60). The local inflammatory environment might also downregulate Treg functionality, and this can be partially mediated by IL-6–producing DCs.58,59
Jouanneau et al.60
reported recently that lysate-loaded DCs are essential for the priming but inefficient for maintaining antitumor immune responses in the GL261 model since late tumor relapses were observed, finally resulting in the cure of only 20% of treated mice. However, we did not observe late tumor relapses. Van Meirvenne et al.61
showed elegantly that in vivo depletion of Tregs enhanced both the primary and memory cytotoxic T-lymphocyte response elicited by mRNA-loaded DCs in an ovalbumin-specific tumor model. In our hands, DCm-GL261-RNA vaccination alone was sufficient for the induction of immunological protection and memory in about half of the mice, while all of the aCD25-treated mice survived but were not protected upon rechallenge. Hence, in the aforementioned setting, our data support a combined immunotherapeutic treatment consisting of Treg depletion and DCm-GL261-RNA vaccination for the induction of an optimal antitumor immune response.
Since systemic monitoring of immune reactions within the brain represents an artificial readout that can only partially reflect local events, we opted for genuine in situ investigation of brain-infiltrating cells. Taken together, we observed that both CD25 depletion and DCm-GL261-RNA vaccination, as well as combined treatment, before challenge with GL261 tumor cells allowed a massive lymphocyte infiltration into the brain. Effector lymphocytes, encompassing both CD4+CD25− and CD4+CD25+FoxP3− and CD8+ T-cells, were upregulated by both aCD25 treatment and vaccination, as well as combined treatment. Combined treatment clearly expanded the Treg population, which is in concordance with the above-mentioned systemic monitoring data. In this regard, time kinetics could provide useful information regarding the expansion and/or reduction in infiltrating lymphocytes, but this was beyond the scope of the present report. Here, we provide evidence that the CD11b− lymphocyte fraction of the brain-infiltrating cells, in particular from animals in which CD25+ cells were prophylactically eliminated, contained the real cytotoxic effector cells, whereas the CD11b+ myeloid cells had little or no cytotoxic effect.
Mice that were treated with either DCm-GL261-RNA alone or DCm-GL261-RNA together with aCD25 clearly displayed a higher number of CD8+CD62Llo lymphocytes, whereas CD4+CD62Llo lymphocytes were mainly increased by aCD25 and combined treatment. This shift toward induction of immunological memory correlates with the initially vaccinated mice being partially protected against IC rechallenge, even without CD25 depletion. The absolute increase in infiltrating CD8+ lymphocytes by DCm-GL261-RNA treatment and the concomitant memorylike phenotype of these cells further illustrate their involvement in the vaccine-mediated antitumor immune response, as already evidenced by previously mentioned in vivo CD8 codepletion experiments.
Overall, our primary goal is to improve glioma immunotherapy both by generating a more effective cellular-mediated antitumor response and by counteracting immune-suppressive mechanisms such as endogenous or tumor-induced Tregs. With the data presented here, we demonstrate the feasibility and efficiency of DC loading with total tumor RNA in vitro as well as in vivo. Moreover, we have partially unraveled the involvement of CD8+
effector T-cells and CD25+
Tregs in the experimental GL261 murine glioma model. Elimination of Tregs by aCD25 treatment or other interventions seems to represent a powerful weapon in the fight against cancer, but unfortunately it is not the ultimate tool in cancer immunotherapy after all.62–65
One should keep in mind that prolonged depletion of Tregs is not feasible due to the risk of eliciting autoimmune disease. Translated to the human system, pilot data on the use of Ontak (denileukin diftitox) and cyclophosphamide in cancer immunotherapy seemed very promising in downregulating Tregs, although conflicting data are arising.66–68
Moreover, not only are Tregs eliminated by aCD25 treatment, but also IL-2–dependent CD4+
helper T-cells and/or proliferating CD8+
lymphocytes can be affected.34
CTLA-4, a negative regulator of endogenous and vaccine-induced antitumor immunity, represents another good selective target in cancer immunotherapy. It has been shown in metastatic melanoma patients that sequential infusions of anti-CTLA-4 mAbs after DC vaccination generate a clinically meaningful antitumor immunity without grade 3 or grade 4 toxicity.69
The prognosis for patients diagnosed with high-grade malignant brain tumors and glioblastoma multiforme (GBM) in particular remains dismal, with a median survival of only 14 months for the latter subcategory. In general, patients respond poorly to the current state-of-the-art treatment strategies, consisting of maximal safe surgical resection, radiotherapy, and chemotherapy.70
Due to spreading of the tumor cells into surrounding areas of the brain, all GBM patients display tumor relapse and die within 18 months.71
Hence, there is an urgent need for new treatment modalities that are able to specifically eradicate or at least suppress the residual glioma cells without serious adverse effects and with acceptable quality of life. In this perspective, autologous DC therapy (in which monocyte-derived DC are loaded with total lysate from the patients’ own tumors) is under investigation by our research group in two phase I/II clinical trials for newly diagnosed and relapsed GBM. So far, postoperative adjuvant DC vaccination in more than 100 patients with relapsed high-grade glioma yields interesting long-term results, with about 25% 2-year survivors after vaccination, which compares favorably to any large study in recurrent high-grade glioma thus far.15
For newly diagnosed GBM, we are currently assessing the integration of DC vaccination into the conventional postoperative radiochemotherapy regimen.
The main objective of the work presented in this study was better insight into the mechanisms governing the induced immune response by DCm-GL261-RNA against malignant glioma. This work opens perspectives for a further detailed study of the cytokine expression patterns both locally in the tumor microenvironment and systemically in the different experimental treatment groups described here. In conclusion, we postulate from the in vivo data presented here that the combined immunotherapeutic approach targeting both Tregs and effector T-cells leads to optimal immunological protection against malignant glioma. The ultimate goal is to refine immunotherapy directed against this type of malignancy, thereby improving the outcome of patients diagnosed with high-grade glial tumors.