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BACKGROUND. Glioblastoma (GBM) is a fast-growing and aggressive brain tumor. GBM is the most frequent malignant primary brain tumour and it can result in death in three-six months, if untreated. The current standard of care (SOC) therapy consists in maximal safe surgical resection followed by radiation therapy and adjuvant temozolomide (Stupp protocol), with a median overall survival (OS) of 8-10 months. However, more than half of GBM patients die within one year from the diagnosis, and only 5% survive more than 5 years despite aggressive therapies. Research has now shifted additional attention to methods of modulating the innate immune system for the treatment of GBM. Moreover, radiotherapy, that plays a key role in GBM treatment, has the potential to convert immunologically ‘cold’ tumors into ‘hot’ tumors by a combination of distinct mechanisms. Overall, literature data indicate that local radiation produces systemic, immune-mediated anti¬tumour and, potentially, antimetastatic effects. Additionally, the combination of local radiotherapy and immune-modulation can augment local tumour control and cause distant (abscopal) antitumour effects through increased tumour-antigen release and antigen-presenting cell (APC) cross-presentation, improved dendritic-cell (DC) function, and enhanced T cell priming. In order to sort out the immunomodulatory effects of radiotherapy for brain glioma we conducted this project, also in association with immunetherapy. The radiological response has been evaluated as well. METHODS. Radiotherapy treatment in combination with dendritic cell immunotherapy was evaluated. GL261-glioma bearing immune-competent mice were treated by means of RT (3 fractions, 1 fr/day) as exclusive and concomitant immunotherapy (dendritic cells). Two clinical trials were studied as well. The population was GBM patients treated by means of standard therapy plus DC-vaccine therapy. Response assessment of GBM after radio-chemotherapy and during immunotherapy by delayed contrast trams (treatment response assessment maps) was evaluated as well. RESULTS. Survival, CD8+ T, NK cells were significantly and slightly significant different: control vs RT vs RT-IT. We found that activated microglia persists in both tumor and contralateral brain of irradiated mice. Moreover, RT promoted antitumoral M1 polarization and RT contributed to a massive recruitment of Th1 CD4+ T cells; RT and DC combination contributes to a robust infiltrate of CD8+ T cells. CONCLUSION. Our results confirm that RT can modulate the TME creating a specific chemokine gradient involved in T cell homing. RT in combination with IT can induce an anti-tumour systemic long-lasting effector CD8+ T cell response as well as a local infiltration of NK cells and CD8+ T cells. The combinatorial approach seems to be a promising therapy for GBM patients. It might be evaluated trough other clinical trials in order to confirm the preliminary results. BACKGROUND. Glioblastoma (GBM) is a fast-growing and aggressive brain tumor. GBM is the most frequent malignant primary brain tumour and it can result in death in three-six months, if untreated. The current standard of care (SOC) therapy consists in maximal safe surgical resection followed by radiation therapy and adjuvant temozolomide (Stupp protocol), with a median overall survival (OS) of 8-10 months. However, more than half of GBM patients die within one year from the diagnosis, and only 5% survive more than 5 years despite aggressive therapies. Research has now shifted additional attention to methods of modulating the innate immune system for the treatment of GBM. Moreover, radiotherapy, that plays a key role in GBM treatment, has the potential to convert immunologically ‘cold’ tumors into ‘hot’ tumors by a combination of distinct mechanisms. Overall, literature data indicate that local radiation produces systemic, immune-mediated anti¬tumour and, potentially, antimetastatic effects. Additionally, the combination of local radiotherapy and immune-modulation can augment local tumour control and cause distant (abscopal) antitumour effects through increased tumour-antigen release and antigen-presenting cell (APC) cross-presentation, improved dendritic-cell (DC) function, and enhanced T cell priming. In order to sort out the immunomodulatory effects of radiotherapy for brain glioma we conducted this project, also in association with immunetherapy. The radiological response has been evaluated as well. METHODS. Radiotherapy treatment in combination with dendritic cell immunotherapy was evaluated. GL261-glioma bearing immune-competent mice were treated by means of RT (3 fractions, 1 fr/day) as exclusive and concomitant immunotherapy (dendritic cells). Two clinical trials were studied as well. The population was GBM patients treated by means of standard therapy plus DC-vaccine therapy. Response assessment of GBM after radio-chemotherapy and during immunotherapy by delayed contrast trams (treatment response assessment maps) was evaluated as well. RESULTS. Survival, CD8+ T, NK cells were significantly and slightly significant different: control vs RT vs RT-IT. We found that activated microglia persists in both tumor and contralateral brain of irradiated mice. Moreover, RT promoted antitumoral M1 polarization and RT contributed to a massive recruitment of Th1 CD4+ T cells; RT and DC combination contributes to a robust infiltrate of CD8+ T cells. CONCLUSION. Our results confirm that RT can modulate the TME creating a specific chemokine gradient involved in T cell homing. RT in combination with IT can induce an anti-tumour systemic long-lasting effector CD8+ T cell response as well as a local infiltration of NK cells and CD8+ T cells. The combinatorial approach seems to be a promising therapy for GBM patients. It might be evaluated trough other clinical trials in order to confirm the preliminary results.

Background Pin1 has previously been described to regulate cells that participate in both innate and adaptive immunity. Thus far, however, no role for Pin1 has been described in modulating conventional dendritic cells, innate antigen presenting cells that potently activate naïve T cells, thereby bridging innate and adaptive immune responses. Methodology/Principal Findings When challenged with LPS, Pin1-null mice failed to accumulate spleen conventional dendritic cells (cDC). Analysis of steady-state spleen DC populations revealed that Pin1-null mice had fewer CD8+ cDC. This defect was recapitulated by culturing Pin1-null bone marrow with the DC-instructive cytokine Flt3 Ligand. Additionally, injection of Flt3 Ligand for 9 days failed to induce robust expansion of CD8+ cDC in Pin1-null mice. Upon infection with Listeria monocytogenes, Pin1-null mice were defective in stimulating proliferation of adoptively transferred WT CD8+ T cells, suggesting that decreases in Pin1 null CD8+ cDC may affect T cell responses to infection in vivo. Finally, upon analyzing expression of proteins involved in DC development, elevated expression of PU.1 was detected in Pin1-null cells, which resulted from an increase in PU.1 protein half-life. Conclusions/Significance We have identified a novel role for Pin1 as a modulator of CD8+ cDC development. Consistent with reduced numbers of CD8+ cDC in Pin1-null mice, we find that the absence of Pin1 impairs CD8+ T cell proliferation in response to infection with Listeria monocytogenes. These data suggest that, via regulation of CD8+ cDC production, Pin1 may serve as an important modulator of adaptive immunity.

CD133 is a hallmark of primitive myeloid progenitors. We have addressed whether human cord blood cells selected for CD133 can generate dendritic cells, and Langerhans cells in particular, in conditions that promote that generation from CD34+ progenitors. Transforming growth factor-β1 (TGF-β1) and anti–TGF-β1 antibody, respectively, were added in some experiments. With TGF-β, monocytoid cells were recognized after 7 days. Immunophenotypically immature dendritic cells were present at day 14. After 4 more days, the cells expressed CD54, CD80, CD83, and CD86 and were potent stimulators in mixed lymphocyte reaction; part of the cells expressed CD1a and langerin, but not Birbeck granules. Without TGF-β, only a small fraction of cells acquired a dendritic shape and expressed the maturation-related antigens, and lymphocytes were poorly stimulated. With anti–TGF-β, the cell growth was greatly hampered, CD54 and langerin were never expressed, and lymphocytes were stimulated weakly. In conclusion, CD133+ progenitors can give rise in vitro, through definite steps, to mature, immunostimulatory dendritic cells with molecular features of Langerhans cells, although without Birbeck granules. Addition of TGF-β1 helps to stimulate cell growth and promotes the acquisition of mature immunophenotypical and functional features. Neither langerin nor Birbeck granules proved indispensable for lymphocyte stimulation.

Spontaneous Ca(2+) oscillations have been observed in a number of excitable and non-excitable cells, but in most cases their biological role remains elusive. In the present study we demonstrate that spontaneous Ca(2+) oscillations occur in immature human monocyte-derived dendritic cells but not in dendritic cells stimulated to undergo maturation with lipopolysaccharide or other toll like-receptor agonists. We investigated the mechanism and role of spontaneous Ca(2+) oscillations in immature dendritic cells and found that they are mediated by the inositol 1,4,5-trisphosphate receptor as they were blocked by pretreatment of cells with the inositol 1,4,5-trisphosphate receptor antagonist Xestospongin C and 2-aminoethoxydiphenylborate. A component of the Ca(2+) signal is also due to influx from the extracellular environment and may be involved in maintaining the level of the intracellular Ca(2+) stores. As to their biological role, our results indicate that they are intimately linked to the "immature" phenotype and are associated with the translocation of the transcription factor NFAT into the nucleus. In fact, once the Ca(2+) oscillations are blocked with 2-aminoethoxydiphenylborate or by treating the cells with lipopolysaccharide, NFAT remains cytoplasmic. The results presented in this report provide novel insights into the physiology of monocyte-derived dendritic cells and into the mechanisms involved in maintaining the cells in the immature stage.

Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin–dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4−/− DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival.

Several glycoconjugates are involved in the immune response. Sialic acid is frequently the glycan terminal sugar and it may modulate immune interactions. Dendritic cells (DCs) are antigen-presenting cells with high endocytic capacity and a central role in immune regulation. On this basis, DCs derived from monocytes (mo-DC) are utilised in immunotherapy, though many features are ignored and their use is still limited. We analyzed the surface sialylated glycans expressed during human mo-DC generation. This was monitored by lectin binding and analysis of sialyltransferases (ST) at the mRNA level and by specific enzymatic assays. We showed that alpha 2-3-sialylated O-glycans and alpha 2-6- and alpha 2-3-sialylated N-glycans are present in monocytes and their expression increases during mo-DC differentiation. Three main ST genes are committed with this rearrangement: ST6Gal1 is specifically involved in the augmented alpha 2-6-sialylated N-glycans; ST3Gal1 contributes for the alpha2-3-sialylation of O-glycans, particularly T antigens; and ST3Gal4 may contribute for the increased alpha2-3-sialylated N-glycans. Upon mo-DC maturation, ST6Gal1 and ST3Gal4 are downregulated and ST3Gal1 is altered in a stimulus-dependent manner. We also observed that removing surface sialic acid of immature mo-DC by neuraminidase significantly decreased its endocytic capacity, while it increased in monocytes. Our results indicate the STs expression modulates the increased expression of surface sialylated structures during mo-DC generation, which is probably related with changes in cell mechanisms. The ST downregulation after mo-DC maturation probably results in a decreased sialylation or sialylated glycoconjugates involved in the endocytosis, contributing to the downregulation of one or more antigen-uptake mechanisms specific of mo-DC.

SUMMARY We have investigated the occurrence, immunohistochemical profile, ultrastructural features and relationships to lymphocytes of the non-lymphoid accessory cells in the dermal infiltrate of five patients affected by B celt lymphoma with secondary involvement of the skin. Typical nonlymphoid accessory cells were found in all cases. Most of these cells had ultrastructural features which resembled those of the poorly differentiated dendritic reticulum cells described in follicular lymphomas of the lymph nodes. The immunohistochemical findings of DRC-1 +, C3b r+ dendritic cells often arranged in follicular-like structures with neoplastic B cells and only few, scattered OKM1+, OKM5+ mononuclear phagocytes support the hypothesis that the vast majority of the non-lymphoid cells observed in our cases were poorly differentiated dendritic reliculum cells. These results and previously published reports indicate that the organization of the dermal infiltrate of B cell lymphomas tends to reproduce the typical arrangement of the B zone of the lymphoid tissue, although with a lesser degree of differentiation, similar to that observed in lymph node follicular lymphomas.