Generation of tumor-reactive CTL against the tumor-associated antigen HER2 using retrovirally transduced dendritic cells derived from CD34+ hemopoietic progenitor cells.

Ag-specific CD8+ CTL are crucial for effective tumor rejection. Attempts to treat human malignancies by adoptive transfer of tumor-reactive CTL have been limited due to the difficulty of generating and expanding autologous CTL with defined Ag specificity. The current study examined whether human CTL can be generated against the tumor-associated Ag HER2 using autologous dendritic cells (DC) that had been genetically engineered to express HER2. DC progenitors were expanded by culturing CD34+ hemopoietic progenitor cells in the presence of the designer cytokine HyperIL-6. Proliferating precursor cells were infected by a retroviral vector encoding the HER2 Ag and further differentiated into CD83+ DC expressing high levels of MHC, adhesion, and costimulatory molecules. Retroviral transduction of DC resulted in the expression of the HER2 molecule with a transduction efficiency of 15%. HER2-transduced DC correctly processed and presented the Ag, because HLA-A*0201-positive DC served as targets for CTL recognizing the HLA-A*0201-binding immunodominant peptide HER2(369-377). HER2-transduced DC were used as professional APCs for stimulating autologous T lymphocytes. Following repetitive stimulation, a HER2-specific, HLA-A*0201-restricted CTL line was generated that was capable of lysing HLA-A*0201-matched tumor cells overexpressing HER2. A CD8+ T cell clone could be generated that displayed the same specificity pattern as the parenteral CTL line. The ability to generate and expand HER2-specific, MHC class I-restricted CTL clones using HER2-transduced autologous DC in vitro facilitates the development of adoptive T cell transfer for patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.

Resistance to Trypanosoma cruzi infection in mice does not necessarily correlate with production of interferon-gamma in vivo.

Interferon-gamma (IFN-gamma) is the most important mediator of inhibition of intracellular replication of Trypanosoma cruzi in vitro and has a protective effect against this parasite if administered in vivo. Here we have analyzed the importance of IFN-gamma for resistance against a lethal infection with T. cruzi in a mouse model system. Resistant B6D2 mice survived the infection with a virulent strain of T. cruzi, whereas susceptible BALB/c mice died within 3 weeks. Both strains produced large amounts of IFN-gamma after infection. Surprisingly, susceptible mice had higher serum concentrations of IFN-gamma and showed, using in situ hybridization a stronger increase in IFN-gamma mRNA-producing cells in their spleens than resistant mice. Moreover, this pattern was also found when immune spleen cells were stimulated with parasite antigens in vitro. However, a marked difference between these mice was found in the production of IL-4, which was much higher in susceptible mice in vivo and in vitro. No difference was found for IL-10. These data show that, at least in the mouse strain/parasite combination used, production of IFN-gamma is not the decisive factor determining resistance or susceptibility to T. cruzi. Rather, it is possible that the balance between protective (e.g., IFN-gamma) and exacerbative cytokines (e.g., IL-4) may decide over disease control or progression.

Trypanosoma cruzi induces strong IL-12 and IL-18 gene expression in vivo: correlation with interferon-gamma (IFN-gamma) production.

IFN-gamma, produced after infection with Trypanosoma cruzi, has been shown to be crucial in the determination of resistance or susceptibility. We have performed a detailed study on the expression of IFN-gamma and of the IFN-gamma-inducing cytokines IL-12 and IFN-gamma-inducing factor (IGIF)/IL-18 with regard to time course and tissue localization. IFN-gamma was present in high amounts in the serum and in the supernatants of unseparated spleen cells and isolated CD4+ and CD8+ T cells from the spleens of infected mice which were stimulated ex vivo with T. cruzi. Using the in situ hybridization technique we demonstrate that IL-12 p40 messages were expressed in the spleen and increased during infection, correlating with the expression of IFN-gamma transcripts. Furthermore, we show for the first time that the mRNA for the cytokine IL-18 was induced by a parasitic infection and that this expression increased during infection with T. cruzi. Interestingly, the message for IL-18 was produced earlier during infection and already had declined until day 38, when IFN-gamma and IL-12 p40 transcripts were optimally expressed. Surprisingly, the changes in IL-12 and IL-18 mRNA production were clearly seen only by in situ hybridization, but less clearly by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). This is possibly due to the extensive activation and proliferation of spleen cells observed during infection leading to a dilution of these specific mRNAs.