Naturally occurring short splice variant of CYLD positively regulates dendritic cell function.

Deubiquitination of NF-kappaB members by CYLD is crucial in controlling the magnitude and nature of cell activation. The role of the naturally occurring CYLD splice variant in dendritic cell (DC) function was analyzed using CYLD(ex7/8) mice, which lack the full-length CYLD (flCYLD) transcript and overexpress the short splice variant (sCYLD). Bone marrow-derived DCs from CYLD(ex7/8) mice display a hyperactive phenotype in vitro and in vivo and have a defect in establishing tolerance with the use of DEC-205-mediated antigen targeting to resting DCs. The combination of sCYLD overexpression and lack of flCYLD in CYLD(ex7/8) DCs leads to enhanced NF-kappaB activity accompanied by an increased nuclear translocation of the IkappaB molecule Bcl-3, along with nuclear p50 and p65. This suggests that, in contrast to flCYLD, sCYLD is a positive regulator of NF-kappaB activity, and its overexpression induces a hyperactive phenotype in DCs.

Heat shock protein-antigen fusions lose their enhanced immunostimulatory capacity after endotoxin depletion.

Heat shock proteins (HSPs) induce cross-presentation of antigens by dendritic cells (DC) as well as DC maturation. These properties make HSP antigen complexes good candidates to prime CD8 T cell responses against tumor-associated antigens. In this study, we analyzed four different members of the HSP70 family fused to a fragment of ovalbumin (OVA) as a model tumor antigen. E. coli-derived recombinant HSP70-OVA fusion proteins efficiently primed antigen-specific cytotoxic T cells in short-term in vivo immunization assays. Because of concerns that the adjuvant effect of HSPs may be due to endotoxin contamination, we studied this issue in detail. Induction of OVA-specific cytotoxicity was significantly decreased in mice deficient for the LPS receptor, TLR4. After careful removal of endotoxins, immunization with HSP70-OVA failed to prime cytotoxic T cell responses. However, we obtained strong in vivo kill responses when endotoxin-depleted HSP70-OVA was used in combination with the TLR9 ligand CpG oligodeoxynucleotide 1668. Importantly, prophylactic and therapeutic treatment with endotoxin-depleted HSP70-OVA together with CpG significantly delayed the outgrowth of OVA-expressing B16 melanoma cells. However, we were unable to detect significant differences in the magnitudes of immune responses against endotoxin-depleted recombinant OVA vs. endotoxin-depleted HSP70-OVA fusion protein. Thus, immunization with recombinant HSP70-antigen fusion protein does not provide an advantage over recombinant antigen alone when combined with a suitable adjuvant. Altogether, our data suggest that the adjuvant effect of the HSP70 part of the fusion protein is completely lost after endotoxin removal.

A critical role for TNF receptor-associated factor 1 and Bim down-regulation in CD8 memory T cell survival.

The mechanisms that allow the maintenance of immunological memory remain incompletely defined. Here we report that tumor necrosis factor receptor (TNFR)-associated factor (TRAF) 1, a protein recruited in response to several costimulatory TNFR family members, is required for maximal CD8 T cell responses to influenza virus in mice. Decreased recovery of CD8 T cells in vivo occurred under conditions where cell division was unimpaired. In vitro, TRAF1-deficient, antigen-activated T cells accumulated higher levels of the proapoptotic BH3-only family member Bim, particularly the most toxic isoform, Bim(S). In the presence of excess IL-15, memory phenotype T cells with similar surface phenotype and comparable levels of Bcl-2 family members could be generated from WT or TRAF1-deficient T cell receptor transgenic OT-I T cells. However, when the memory CD8 T cells were allowed to compete for survival signals in the absence of antigen in vivo, the TRAF1-deficient T cells showed decreased recovery compared with TRAF1-sufficient T cells. This defect in T cell recovery in vivo was alleviated by introduction of siRNA to down-modulate Bim in TRAF1-deficient memory T cells. These studies identify the TRAF1 signaling axis and Bim down-regulation as critical for CD8 memory T cell survival in vivo.