A role for the Hsp90 molecular chaperone family in antigen presentation to T lymphocytes via major histocompatibility complex class II molecules

The heat shock protein (HSP) Hsp90 is known to chaperone cytosolic peptides for MHC class I (MHCI)-restricted antigen presentation to T lymphocytes. We now demonstrate a role for Hsp90 activity in presentation of antigens on MHCII. Treatment of mouse antigen-presenting cells (APC) with the pharmacological Hsp90 inhibitor, geldanamycin, inhibited MHCII-mediated presentation of endocytosed and cytosolic proteins as well as synthetic peptides to specific T cells. Ectopic expression of human Hsp90 in APC enhanced MHCII-mediated antigen presentation. Further, pharmacological Hsp90 inhibition reduced, while retroviral Hsp90 overexpression enhanced, the levels of stable compact MHCII heterodimers correlating with the antigen presentation phenotype. Pharmacological inhibition of Hsp90 activity in IFN-γ-treated APC resulted in severe abrogation of MHCII-restricted presentation of cytosolic antigen, but only partially inhibited exogenous antigen presentation. Our data suggest a major role for Hsp90 activity in MHCII-mediated antigen presentation pathways, and implicate IFN-γ-inducible Hsp90-independent mechanisms

MHC class I-restricted presentation of maleylated protein binding to scavenger receptors

Pathways for loading exogenous protein-derived peptides on MHC class I are thought to be present mainly in monocyte-lineage cells and to involve phagocytosis- or macropinocytosis-mediated antigenic leakage into either cytosol or extracellular milieu to give peptide access to MHC class I. We show that maleylation of OVA enhanced its presentation to an OVA-specific MHC class I-restricted T cell line by both macrophages and B cells. This enhanced presentation involved uptake through receptors of scavenger receptor (SR)-like ligand specificity, was TAP-1-independent, and was inhibited by low levels (2 mM) of ammonium chloride. No peptide loading of bystander APCs by maleylated (maleyl) OVA-pulsed macrophages was detected. Demaleylated maleyl-OVA showed enhanced MHC class I-restricted presentation through receptor-mediated uptake and remained highly sensitive to 2 mM ammonium chloride. However, if receptor binding of maleyl-OVA was inhibited by maleylated BSA, the residual presentation was relatively resistant to 2 mM ammonium chloride. Maleyl-OVA directly introduced into the cytosol via osmotic lysis of pinosomes was poorly presented, confirming that receptor-mediated presentation of exogenous maleyl-OVA was unlikely to involve a cytosolic pathway. Demaleylated maleyl-OVA was well presented as a cytosolic Ag, consistent with the dependence of cytosolic processing on protein ubiquitination. Thus, receptor-specific delivery of exogenous protein Ags to APCs can result in enhanced MHC class I-restricted presentation, suggesting that the exogenous pathway of peptide loading for MHC class I may be a constitutive property dependent mainly on the quantity of Ag taken up by APCs

Disruption of T cell tolerance to self-immunoglobulin causes polyclonal B cell stimulation followed by inactivation of responding autoreactive T cells

Scavenger receptor (SR)-specific delivery by maleylation of a ubiquitous self-protein, Ig, to SR-bearing APCs results in self-limiting induction of autoimmune effects in vivo. Immunization with maleyl-Ig breaks T cell tolerance to self-Ig and causes hypergammaglobulinemia, with increases in spleen weight and cellularity. The majority of splenic B cells show an activated phenotype upon maleyl-Ig immunization, leading to large-scale conversion to a CD138+ phenotype and to significant increases in CD138-expressing splenic plasma cells. The polyclonal B cell activation, hypergammaglobulinemia, and autoreactive Ig-specific T cell responses decline over a 2-mo period postimmunization. Following adoptive transfer, T cells from maleyl-Ig-immune mice taken at 2 wk postimmunization can induce hypergammaglobulinemia in the recipients, but those taken at 10 wk postimmunization cannot. Hypergammaglobulinemia in the adoptive transfer recipients is also transient and is followed by an inability to respond to fresh maleyl-Ig immunization, suggesting that the autoreactive Ig-specific T cells are inactivated peripherally following disruption of tolerance. Thus, although autoreactive T cell responses to a ubiquitous self-Ag, Ig, are induced by SR-mediated delivery to professional APCs in vivo resulting in autoimmune pathophysiological effects, they are effectively and rapidly turned off by inactivation of these activated Ig-specific T cells in vivo

HIV-1 Nef induces a Rab11-dependent routing of endocytosed immune costimulatory proteins CD80 and CD86 to the Golgi

The Nef protein of HIV-1 removes the immune costimulatory proteins CD80 and CD86 from the cell surface by a unique clathrin- and dynamin-independent, actin-based endocytic pathway that deploys coupled activation of c-src and Rac. In this study, we show that, similar to major histocompatibility complex class I (MHCI), Nef subsequently reroutes CD80 and CD86 to the Golgi region. However, not only are CD80/CD86 internalized by a different mechanism from MHCI but also the vesicular pathway of Golgi delivery for CD80/CD86 is distinct from that employed for MHCI. While MHCI passes through early endosomal and sorting compartments marked by Rab5/early embryonic antigen 1 and ADP ribosylation factor 6, respectively, CD80 and CD86 enter endocytic vesicles that do not acquire conventional early endosomal markers but remain accessible to fluid probes. Rather than being delivered to preexisting Rab11-positive recycling compartments, these vesicles recruit Rab11 de novo. Rab11 activity is also necessary for the delivery of CD80/CD86 in these transitional vesicles to the Golgi region. These data reveal an unusual pathway of endocytic vesicular traffic to the Golgi and its recruitment in a viral immune evasion strategy

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

Differential Regulation of Two Arms of mTORC1 Pathway Fine-Tunes Global Protein Synthesis in Resting B Lymphocytes

Protein synthesis is tightly regulated by both gene-specific and global mechanisms to match the metabolic and proliferative demands of the cell. While the regulation of global protein synthesis in response to mitogen or stress signals is relatively well understood in multiple experimental systems, how different cell types fine-tune their basal protein synthesis rate is not known. In a previous study, we showed that resting B and T lymphocytes exhibit dramatic differences in their metabolic profile, with implications for their post-activation function. Here, we show that resting B cells, despite being quiescent, exhibit increased protein synthesis in vivo as well as ex vivo. The increased protein synthesis in B cells is driven by mTORC1, which exhibits an intermediate level of activation in these cells when compared with resting T cells and activated B cells. A comparative analysis of the transcriptome and translatome of these cells indicates that the genes encoding the MHC Class II molecules and their chaperone CD74 are highly translated in B cells. These data suggest that the translatome of B cells shows enrichment for genes associated with antigen processing and presentation. Even though the B cells exhibit higher mTORC1 levels, they prevent the translational activation of TOP mRNAs, which are mostly constituted by ribosomal proteins and other translation factors, by upregulating 4EBP1 levels. This mechanism may keep the protein synthesis machinery under check while enabling higher levels of translation in B cells

HIV-1 Nef promotes endocytosis of cell surface MHC class II molecules via a constitutive pathway

HIV-1 Nef has been reported to disrupt MHC class II (MHCII)-mediated Ag presentation by a dual strategy that comprises a reduction in cell surface levels of peptide-loaded mature MHCII molecules and a up-regulation of immature MHCII molecules. We show that Nef achieves relocation of MHCII away from the cell surface in monocytic cells by both delaying its transport to the cell surface and by accelerating endocytic removal of cell surface MHCII to a lysosomal compartment. Nef-induced MHCII endocytosis is cholesterol-sensitive but clathrin- and dynamin-independent. Internalized MHCII molecules traverse the early endosomal system and colocalize with pinocytic cargo before reaching lysosomes. Nef-triggered MHCII endocytosis requires Rab5 activity and lyst function, whereas lysosomal trafficking of internalized MHCII molecules requires Rab7 activity. We further show that a similar pathway can remove peptide-MHCII complexes from the surface of monocytic cells not expressing Nef. Our data suggest that Nef uses mechanisms involved in normal MHCII recycling and turnover to mediate the delivery of cell surface MHCII to a lysosomal destination. Thus, Nef-mediated endocytosis of MHCII provides a novel perspective on the regulation of normal MHCII trafficking

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

Direct evidence for a functional role of HLA-DRB1 and -DRB3 gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T lymphocytes

The contribution of the HLA-DRB1, -B3, and -BS gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T cells Isolated from an atopic individual (HLA-DRw12, DR7; DRw52b) with perennial rhinitis was investigated. Using a panel of histocompatlble and histoincompatible accessory cells, the restriction specificity obtained for a long term T cell suggested that a component of the dust mite reactive repertoire recognized antigen in association with DRB3 gene products. Ollgonucleotide DNA typing of the presenting cell panel demonstrated a correlation between the DRw52b allele and T cell responsiveness. Murine fibroblasts expressing DRw52b, but not DRw52a or -c molecules, presented antigen to both the T cell line and cloned T cells (DE26) derived from the line, Indicating that the supertypic specificity DRw52b was able to restrict recognition of dust mite antigens. Additional T cell clones (DE9 and DE41) also isolated from the line were restricted by the products of the B1 gene locus (DRw12B1) as determined by murine fibroblasts transfected with the appropriate HLA-DR genes. Clone DE9 was degenerate in Its restriction specificity, also recognizing dust mite presented by accessory cells expressing the DR2 subtypes. Presentation by fibroblasts transfected with DRw12B1, DR2Dw2B5 genes and EBV-transformed B cell lines expressing DR2Dw21B1 and -B5 indicated that the functional site restricting recognition may be associated with residues 70 and 71 of the DR/3 chain helical wall of the antigen combining site. Furthermore, we have recently demonstrated that both T cell clones DE9 and DE26 induce allergen dependent IgE synthesis in vitro. Thus these results demonstrate directly that the DRB1, -B3, and -B5 gene products are functional in the restriction of T cell recognition of dust mite antigen