C3d adjuvant effects are mediated through the activation of C3d-specific autoreactive T cells.

Complement fragment C3d covalently attached to antigens enhances immune responses, particularly for antigens lacking T-cell epitopes. Enhancement has been attributed to receptor cross-linking between complement receptor CR2 (CD21) and polysaccharide antigen to surface IgM on naïve B cells. Paradoxically, C3d has still been shown to increase immune responses in CD21 knockout mice, suggesting that an auxiliary activation pathway exists. In prior studies, we demonstrated the CD21-independent C3d adjuvant effect might be due to T-cell recognition of C3d T-helper epitopes processed and presented by major histocompatibility complex class II on the B-cell surface. C3d peptide sequences containing concentrated clusters of putative human C3 T-cell epitopes were identified using the epitope-mapping algorithm, EpiMatrix. These peptide sequences were synthesized and shown in vitro to bind multiple human leukocyte antigen (HLA)-DR alleles with high affinity, and induce interferon-γ responses in healthy donor peripheral blood mononuclear cells. In the present studies, we establish further correlations between HLA binding and HLA-specific lymphocyte reactions with select epitope clusters. In addition, we show that the T-cell phenotype of C3d-specific reactive T cells is CD4(+)CD45RO(+) memory T cells. Finally, mutation of a single T-cell epitope residing within the P28 peptide segment of C3d resulted in significantly diminished adjuvant activity in BALB/c mice. Collectively, these studies support the hypothesis that the paradoxical enhancement of immune responses by C3d in the absence of CD21 is due to internalization and processing of C3d into peptides that activate autoreactive CD4(+) T-helper cells in the context of HLA class II.

Recombinant expression, biophysical characterization, and cardiolipin-induced changes of two Caenorhabditis elegans cytochrome c proteins.

Cytochrome c (cyt c) is one of the most widely studied biomolecules, but not much is known about this protein from nematodes. Recombinant expression of Caenorhabditis elegans CYC-2.1 and CYC-2.2 allowed for detailed characterization of their structural features, redox properties, stabilities, and interactions with cardiolipin (CL)-containing liposomes. Using a variety of spectroscopic tools, we show that CYC-2.1 and CYC-2.2 adopt a globular α-helical fold with His/Met heme ligation. The longer CYC-2.2 has a lower thermodynamic stability than CYC-2.1 and lacks His residues to misligate to the heme in the protein's denatured state. Both C. elegans proteins bind to CL-containing liposomes, and these interactions promote the proteins' peroxidase activity but to a much greater degree for CYC-2.2. Dye-to-heme distance distributions from time-resolved fluorescence resonance energy transfer in bimane-labeled CYC-2.1 and CYC-2.2 revealed similar populations of extended and compact conformers for CL-bound proteins, suggesting that their distinct peroxidase activities in the presence of CL arise from differences in the local heme environments for the two polypeptide ensembles. Without inhibition from His misligation, a less stable and more prone to unfolding CYC-2.2 allows for better access of substrates to the heme and thus exhibits higher peroxidase activity. Similar features of the conformational ensembles of CYC-2.1 and CYC-2.2 to those of mammalian cyt c suggest that C. elegans proteins, particularly the former, could serve as useful models for examining the mechanism of cyt c-CL interactions in live organisms.