Rapid T cell-based identification of human tumor tissue antigens by automated two-dimensional protein fractionation.

Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individual cancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4+ Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8+ T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4+ and CD8+ T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele.

Combination of thalidomide and cisplatin in an head and neck squamous cell carcinomas model results in an enhanced antiangiogenic activity in vitro and in vivo.

Thalidomide is an immunomodulatory, antiangiogenic drug. Although there is evidence that it might be more effective in combination with chemotherapy the exact mechanism of action is unclear. Therefore, we investigated its effect in combination with metronomically applied cisplatin in a xenotransplant mouse model characteristic for advanced head and neck squamous cell carcinomas, its possible synergistic action in vitro, and which tumor-derived factors might be targeted by thalidomide. Although thalidomide alone was ineffective, a combined treatment with low-dose cisplatin inhibited significant tumor growth, proliferation and angiogenesis in vivo as well as migration and tube formation of endothelial cells in vitro. Noteworthy, the latter effect was enhanced after coapplication of cisplatin in nontoxic doses. An inhibitory effect on tumor cell migration was also observed suggesting a direct antitumor effect. Although thalidomide alone did not influence cell proliferation, it augmented antiproliferative response after cisplatin application emphasizing the idea of a potentiated effect when both drugs are combined. Furthermore, we could show that antiangiogenic effects of thalidomide are related to tumor-cell derived factors including vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor and Il-8 some known and with, granulocyte colony stimulating growth factor and granulocyte macrophage colony stimulating growth factor, some new target molecules of thalidomide. Altogether, our findings reveal new insights into thalidomide-mediated antitumor and antiangiogenic effects and its interaction with cytostatic drugs.

Immunomodulatory derivative of thalidomide (IMiD CC-4047) induces a shift in lineage commitment by suppressing erythropoiesis and promoting myelopoiesis.

Immunomodulatory derivative (IMiD) CC-4047, a new analog of thalidomide, directly inhibits growth of B-cell malignancies in vivo and in vitro and exhibits stronger antiangiogenic activity than thalidomide. However, there is little information on whether CC-4047 affects normal hematopoiesis. Here we investigated the effect of CC-4047 on lineage commitment and differentiation of hematopoietic stem cells. We found that CC-4047 effectively inhibits erythroid cell colony formation from CD34+ cells and increases the frequency of myeloid colonies. We also demonstrate that development of both erythropoietin-independent and erythropoietin-dependent red cell progenitors was strongly inhibited by CC-4047, while terminal red cell differentiation was unaffected. DNA microarray analysis revealed that red cell transcription factors, including GATA-1, GATA-2, erythroid Kruppel-like factor (EKLF), and growth factor independence-1B (Gfi-1b), were down-regulated in CC-4047-treated CD34+ cells, while myeloid transcription factors such as CCAAT/enhancer binding protein-alpha (C/EBPalpha), C/EBPdelta, and C/EBPepsilon were induced. Analysis of cytokine secretion indicated that CC-4047 induced secretion of cytokines that enhance myelopoiesis and inhibit erythropoiesis. In conclusion, these data indicate that CC-4047 might directly influence lineage commitment of hematopoietic cells by increasing the propensity of stem and/or progenitor cells to undergo myeloid cell development and concomitantly inhibiting red cell development. Therefore, CC-4047 provides a valuable tool to study the mechanisms underlying lineage commitment.