Investigation of immunosuppressive mechanisms in a mouse glioma model.

The development of an immune competent mouse model for the study of immunosuppressive mechanisms is important for improving the efficacy of brain tumor immunotherapy. In the present study we investigated regulatory T cells (Tregs), TGF-beta1 and other putative immunosuppressive cytokines using GL261 mouse glioma in C57BL mice. We explored whether tumor growth factor-beta1 (TGF-beta1) is expressed and secreted by glioma cells constitutively or in response to a T-cell mediated immunity (simulated by conditioned media from T cells (TCM) activated by anti-CD3 antibody). We also investigated TGF-beta1's role in Treg mediated immunosuppression by quantifying TGF-beta1secretion from T regulatory cells (Tregs) co-incubated with GL261 cells as compared to Tregs alone. Finally, we studied other newly identified cytokines that were secreted preferentially by glioma cells in response to CD3 activated TCM versus cytokines secreted by glioma cells in absence of T-cell activation (naïve TCM). TGF-beta1expression was studied using RT-PCR and secretion was quantified using ELISA. A 308 protein cytokine array was used to identify and quantify cytokine expression. TGF-beta1expression and secretion from glioma cells was found to be up-regulated by conditioned media from CD3-activated T cells, suggesting that this immunosuppressive cytokine is not secreted constitutively but in response to immunity. TGF-beta1 was not found to be differentially secreted by Tregs co-incubated with glioma cells as compared to Tregs alone. This data suggest that TGF-beta1immunosupppression may not be a Treg dependent mechanism in this glioma model. Finally, the cytokine array elucidated several other cytokines which were up-regulated or down-regulated by CD3-activated TCM. These results have several implications for enhancing immunotherapy treatment, including the potential benefit of TGF-beta1inhibition in conjunction with immunotherapy, as well as the illumination of several other potential cytokine targets to be explored as shown by the cytokine array.

Regulation of melanoma epitope-specific cytolytic T lymphocyte response by immature and activated dendritic cells, in vitro.

Dendritic cell (DC)-based immunization in cancer has proven to be a promising approach. However, just as DCs are crucial accessory cells in generating immune responses, they also seem to participate in tolerance induction, especially against peripheral "self" antigens. The bulk of the evidence that DCs present peripheral self antigens to induce tolerance has, however, come mostly from studies in transgenic animal models. A tolerogenic function of DCs for peripheral self antigens in a human model has not been critically examined. In this study using the Melan-A/MART-1(27-35) peptide as a model for self but melanoma-associated antigen-against which human hosts often harbor CD8(+) CTL precursors with high frequencies-we confirm that although immature dendritic cells (iDCs) are inefficient antigen presenting cells (APCs), fully activated DCs efficiently activate melanoma epitope-specific CD8(+) CTL precursors, in vitro. We, however, show that in a direct epitope presentation schema, iDCs neither delete nor anergize epitope-specific CD8(+) T cells in primary or secondary stimulation. Interestingly, iDCs and activated DCs can delete a large fraction of the epitope-specific CTLs on tertiary stimulation. The deletion is induced in an epitope-specific manner and through apoptosis. These observations, therefore, have implications on the DC-based cancer vaccine designs and are relevant in the inquiry into the role of DCs on tolerance induction.

Antigen presentation by MART-1 adenovirus-transduced interleukin-10-polarized human monocyte-derived dendritic cells.

Dendritic cells (DC) play critical roles in generating an immune response and in inducing tolerance. Diverse microenvironmental factors can 'polarize' DC toward an immunogenic or non-immunogenic phenotype. Among the various microenvironmental factors, interleukin-10 (IL-10) exhibits a potent immunosuppressive effect on antigen-presenting cells (APC). Here, we show that monocyte-derived DC generated in the presence of IL-10 exhibit a profound down-regulation of many genes that are associated with immune activation and show that the IL-10-grown DC are poor stimulators of CD8(+) T cells in a strictly autologous and major histocompatibility complex (MHC) class I-restricted melanoma antigen recognized by T cells (MART-1) epitope presentation system. However, these IL-10-grown DC can efficiently activate the epitope-specific CD8(+) T cells when they are made to present the epitope following transduction with an adenoviral vector expressing the MART-1 antigen. In addition, we show that the MART-1 protein colocalizes with the MHC class I protein, equally well, in the iDC and in the DC cultured in presence of IL-10 when both DC types are infected with the viral vector. We also show that the vector transduced DC present the MART-1(27-35) epitope for a sustained period compared to the peptide pulsed DC. These data suggest that although DCs generated in the presence of IL-10 tend to be non-immunogenic, they are capable of processing and presenting an antigen when the antigen is synthesized within the DC.