Modulation of T cell cytokine profiles and peptide-MHC complex availability in vivo by delivery to scavenger receptors via antigen maleylation

We have previously shown that conversion of proteins to scavenger receptor (SR) ligands by maleylation increases their immunogenicity. We now show that maleyl-Ag-immune spleen cells make relatively more IFN-ϒ and less IL-4 or IL-10 than native Ag-immune cells. This is also reflected in the IgG1:IgG2a ratios in Abs generated in vivo. SR engagement on macrophages does not alter their surface levels of the adhesive/costimulatory molecules CD11a/CD18, CD11b/CD18, CD24, CD54, or CD40, nor does it enhance their ability to support anti-CD3-driven proliferation of naive T cells in vitro. Costimulatory molecules implicated in differential Th1/Th2 commitment-CD80, CD86, and IL-12-are not inducible by SR ligation. In addition to macrophages and dendritic cells, B cells also show receptor-mediated uptake and enhanced presentation of maleyl-Ags. Using a monoclonal T cell line to detect peptide-MHC complexes expressed on spleen cells in Ag-injected mice, we find that higher levels of these complexes are generated in vivo from maleyl-proteins and they persist longer than those generated from the native protein. Together, these data suggest that in certain situations, the levels of cognate ligand available and/or the time course of their availability may play a major role in determining the cytokine profiles of the responding T cells in addition to the costimulatory signals implicated so far

Efficient presentation of both cytosolic and endogenous transmembrane protein antigens on MHC class II is dependent on cytoplasmic proteolysis

Peptides from extracellular proteins presented on MHC class II are mostly generated and loaded in endolysosomal compartments, but the major pathways responsible for loading peptides from APC-endogenous sources on MHC class II are as yet unclear. In this study, we show that MHC class II molecules present peptides from proteins such as OVA or conalbumin introduced into the cytoplasm by hyperosmotic pinosome lysis, with efficiencies comparable to their presentation via extracellular fluid-phase endocytosis. This cytosolic presentation pathway is sensitive to proteasomal inhibitors, whereas the presentation of exogenous Ags taken up by endocytosis is not. Inhibitors of nonproteasomal cytosolic proteases can also inhibit MHC class II-restricted presentation of cytosolically delivered protein, without inhibiting MHC class I-restricted presentation from the same protein. Cytosolic processing of a soluble fusion protein containing the peptide epitope I-Eα 52-68 yields an epitope that is similar to the one generated during constitutive presentation of I-Eα as an endogenous transmembrane protein, but is subtly different from the one generated in the exogenous pathway. Constitutive MHC class II-mediated presentation of the endogenous transmembrane protein I-Eα is also specifically inhibited over time by inhibitors of cytosolic proteolysis. Thus, Ag processing in the cytoplasm appears to be essential for the efficient presentation of endogenous proteins, even transmembrane ones, on MHC class II, and the proteolytic pathways involved may differ from those used for MHC class I-mediated presentation

Scavenger receptor-specific allergen delivery elicits IFN-&#947;-dominated immunity and directs established T<SUB>H</SUB>2-dominated responses to a nonallergic phenotype

Background: Immunotherapeutic approaches to allergy consist of reliably changing allergen-specific TH2 immunity associated with secretion of IL-4, IL-5, and IL-13, along with IgE antibodies in atopic individuals to TH1 immunity. Our earlier data show that targeting of protein antigens to antigen-presenting cells (APCs), such as macrophages, by means of scavenger receptors (SRs) results in a pronounced TH1 immunity. Here we demonstrate a novel experimental approach for the conversion of TH2 immunity to TH1 immunity by using SR delivery of allergens. Objectives: We sought to show that targeting of allergens by means of SRs to APCs triggers TH1 immunity and that an established TH2 immunity to the Der p 1-immunodominant peptide 111-139 (p1, 111-139) can be modulated to a nonallergic TH1 phenotype. Methods: Analysis of the T cell-derived cytokines IL-4, IL-5, IL-13, and IFN-&#947; in response to p1, 111-139 in C57BL/6 mice 7 to 42 days after immunization, measurement of specific antibody responses, eosinophilic infiltrate, and skin hypersensitivity in response to allergen challenge constitute the parameters of in vivo immunity. Results: We show that p1, 111-139, when delivered to APCs by means of SR, elicits a TH1-dominant immunity. If it is delivered to APCs either after chemical coupling to SR ligands or by means of mere coadsorption on alum in the presence of an SR ligand, the established TH2 immunity can be modified to TH1 immunity. Conclusions: SR-mediated delivery of allergens has immunotherapeutic potential that may be usable in atopic individuals

Structural and Functional Analysis of the Costimulatory Receptor Programmed Death-1

PD-1, a member of the CD28/CTLA-4/ICOS costimulatory receptor family, delivers negative signals that have profound effects on T and B cell immunity. The 2.0 Å crystal structure of the extracellular domain of murine PD-1 reveals an Ig V-type topology with overall similarity to the CTLA-4 monomer; however, there are notable differences in regions relevant to function. Our structural and biophysical data show that PD-1 is monomeric both in solution as well as on cell surface, in contrast to CTLA-4 and other family members that are all disulfide-linked homodimers. Furthermore, our structure-based mutagenesis studies identify the ligand binding surface of PD-1, which displays significant differences compared to those present in the other members of the family