A proteomics strategy for the enrichment of receptor-associated complexes.

Multimeric protein complexes are important for cell function and are being identified by proteomics approaches. Enrichment strategies, such as those employing affinity matrices, are required for the characterization of such complexes, for example, those containing growth factor receptors. The receptor for the macrophage lineage growth factor, macrophage-colony stimulating factor (M-CSF or CSF-1), is the tyrosine kinase, c-Fms. There is evidence that the CSF-1 receptor (CSF-1R) forms distinct multimeric complexes involving autophosphorylated tyrosines in its cytoplasmic region; however, these complexes are difficult to identify by immunoprecipitation, making enrichment necessary. We report here the use of a tyrosine-phosphorylated, GST-fusion construct of the entire CSF-1R cytoplasmic region to characterize proteins putatively associating with the activated CSF-1R. Besides signalling molecules known to associate with the receptor or be involved in CSF-1R-dependent signalling, mass spectrometry identified a number of other molecules binding to the construct. So far among these candidate proteins, dynein, claudin and silencer of death domains co-immunoprecipitated with the CSF-1R, suggesting association. This affinity matrix method, using an entire cytoplasmic region, may have relevance for other growth factor receptors.

Selectively impaired CD8+ but not CD4+ T cell cycle arrest during priming as a consequence of dendritic cell interaction with plasmodium-infected red cells.

Individuals living in malaria-endemic areas show generally low T cell responses to malaria Ags. In this study, we show murine dendritic cell (DC) interaction with parasitized erythrocytes (pRBC) arrested their maturation, resulting in impaired ability to stimulate naive, but not recall T cell responses in vitro and in vivo. Moreover, within the naive T cell population, pRBC-treated DC were selectively deficient in priming CD8(+) but not CD4(+) T cells. Indeed, DC that had taken up pRBC were shown for the first time to efficiently prime CD4(+) T cell responses to a known protective merozoite Ag, MSP4/5. In contrast, impaired priming resulted in decreases in both proliferation and cytokine production by CD8(+) T cells. Deficient priming was observed to both a model and a Plasmodium berghei-specific CD8(+) T cell epitope. The mechanisms underlying the inability of parasite-treated DC to prime CD8(+) T cells were explored. pRBC treatment of DC from wild-type C57BL/6, but not from IL-10 knockout animals, suppressed DC-mediated T cell priming across a Transwell, suggesting active IL-10-dependent suppression. CD8(+) T cells were arrested at the G(0) stage of the cell cycle after two cell divisions post-Ag stimulation. The proliferation arrest was partially reversible by the addition of IL-2 or IL-7 to responder cultures. These results suggest that in malaria-endemic areas, priming of CD8(+) T cell responses may be more difficult to induce via vaccination than the priming of CD4(+) T cells. Moreover, pathogens may selectively target the CD8(+) T cell arm of protective immunity for immune evasion.

Short peptide sequences containing MHC class I and/or class II epitopes linked to nano-beads induce strong immunity and inhibition of growth of antigen-specific tumour challenge in mice.

Peptide based vaccines offer practical advantages, but unmodified peptides usually require an adjuvant or delivery vehicle to promote immunogenicity. When peptides containing ovalbumin (OVA) derived CD4 and CD8 T cell epitopes were conjugated to 0.05 microm nano-beads, they gave strong immune responses and inhibition of growth of tumour cells expressing the CD8 T cell epitope with MHC class I. These responses were inducible with both high (50 microg) and low (5 microg) peptide doses after a single immunisation. The helper CD4 T cell epitope was unnecessary for induction of CD8 T cell or tumour challenge responses. However, the CD4 T cell epitope contained a B cell epitope and triggered strong antibody responses. This simple approach offers a convenient experimental tool and a potentially useful clinical method for peptide immunisation.

Dendritic cells induce immunity and long-lasting protection against blood-stage malaria despite an in vitro parasite-induced maturation defect.

Dendritic cells (DC) suffer a maturation defect following interaction with erythrocytes infected with malaria parasites and become unable to induce protective malaria liver-stage immunity. Here we show that, by contrast, maturation-arrested DC in vitro are capable of the successful induction of antigen-specific gamma interferon (IFN-gamma) and interleukin 4 (IL-4) T-cell responses, antibody responses, and potent protection against lethal blood-stage malaria challenge in vivo. Similar results were found with DC pulsed with intact parasitized Plasmodium yoelii or Plasmodium chabaudi erythrocytes. Cross-strain protection was also induced. High levels of protection (80 to 100%) against lethal challenge were evident from 10 days after a single immunization and maintained up to 120 days. Interestingly, correlation studies versus blood-stage protection at different time points suggest that the immune effector mechanisms associated with protection could change over time. Antibody-independent, T-cell- and IL-12-associated protection was observed early after immunization, followed by antibody and IL-4-associated, IFN-gamma-independent protection in long-term studies. These results indicate that DC, even when clearly susceptible to parasite-induced maturation defect effects in vitro, can be central to the induction of protection against blood-stage malaria in vivo.