Selective addition of CXCR3+CCR4-CD4+ Th1 cells enhances generation of cytotoxic T cells by dendritic cells in vitro

Increasing importance is being given to the stimulation of Th1 response in cancer immunotherapy because its presence can shift the direction of adaptive immune responses toward protective immunity. Based on chemokine receptor expression, CXCR3+CCR4-CD4+ T cells as Th1-type cells were investigated its capacity in monocyte-derived dendritic cell (DC) maturation and polarization, and induction of antigen specific cytotoxic T lymphocytes (CTL) in vitro. The levels of IL-4, IL-5 and IL-10 were decreased to the basal level compared with high production of IFN-gamma, TNF-alpha, and IL-2 in CXCR3+CCR4-CD4+ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Co-incubation of activated CD4+ or CXCR3+CCR4-CD4+ T cells with DC (CD4+/DC or CXCR3+CD4+/DC, respectively) particularly up-regulated IL-12 and CD80 expression compared with DC matured with TNF-alpha and LPS (mDC). Although there was no significant difference between the effects of the CXCR3+CCR4-CD4+ and CD4+ T cells on DC phenotype expression, CXCR3+CD4+/DC in CTL culture were able to expand number of CD8+ T cells and increased frequencies of IFN-gamma secreting cells and overall cytolytic activity against tumor antigen WT-1. These results demonstrated that the selective addition of CXCR3+CCR4-CD4+ T cells to CTL cultures could enhance the induction of CTLs by DC in vitro, and implicated on a novel strategy for adoptive T cell therapy.

Induction of Aspergillus fumigatus Specific T Cells using Dendritic Cells Pulsed with Asp f16 Recombinant Protein in vitro

Invasive aspergillosis is the most common Aspergillus fumigatus infection in immunocompromised patients. Although the treatment of the invasive aspergillosis has been mostly relied on antifungal agents, there still exists the need for more effective therapy. To develop cellular immunotherapy specific for Aspergillus fumigatus, we generated specific T cells using dendritic cells (DCs) pulsed with an Aspergillus fumigatus derived recombinant protein in vitro and examined their functions. The f16. p2+3 region containing the conserved region of Asp f16 gene was cloned from Aspergillus fumigatus and the recombinant protein was produced in E. coli. IFN-gamma secretion from the T cells stimulated with recombinant f16. p2+3 (rf16. p2+3) was measured by enzyme linked immunospot (ELISPOT) assay and the cytolytic activity of the stimulated T cells by 51Cr release assay. The number of IFN-gamma secreting cells were significantly increased in the peripheral blood mononuclear cells (PBMCs) stimulated with the rf16. p2+3 pulsed DCs (31+/-12 spots/10(4) cells), compared to that of PBMCs directly stimulated with rf16. p2+3 (83+/-15 spots/10(6) cells). IFN-gamma ELISPOT assay using purified CD4+ or CD8+ as responder cells showed that CD4+ T cells (43 spots/10(4) cells) mainly produced IFN-gamma compared with CD8+ T cells (7 spots/10(4) cells). Furthermore, helper T cells specific for rf16. p2+3 could be efficiently generated by the stimulation with DCs for two weeks. However, cytotoxic T lymphocyte activity was not induced. Our results suggest that the rf16. p2+3 protein could be used for the generation of helper T cells in vitro.