James R. Drake

Francisella tularensis-Infected Macrophages Release Prostaglandin E2 that Blocks T Cell Proliferation and Promotes a Th2-Like Response

Francisella tularensis is a highly infectious bacterial pathogen, and is likely to have evolved strategies to evade and subvert the host immune response. In this study, we show that F. tularensis infection of macrophages alters T cell responses in vitro, by blocking T cell proliferation and promoting a Th2-like response. We demonstrate that a soluble mediator is responsible for this effect and identify it as PGE2. Supernatants from F. tularensis-infected macrophages inhibited IL-2 secretion from both MHC class I and MHC class II-restricted T cell hybridomas, as well as enhanced a Th2-like response by inducing increased production of IL-5. Furthermore, the soluble mediator blocked proliferation of naive MHC class I-restricted T cells when stimulated with cognate tetramer. Indomethacin treatment partially restored T cell proliferation and lowered IL-5 production to wild-type levels. Macrophages produced PGE2 when infected with F. tularensis, and treatment of infected macrophages with indomethacin, a cyclooxygenase-1/cyclooxygenase-2 inhibitor, blocked PGE2 production. To further demonstrate that PGE2 was responsible for skewing of T cell responses, we infected macrophages from membrane PGE synthase 1 knockout mice (mPGES1−/−) that cannot produce PGE2. Supernatants from F. tularensis-infected membrane PGE synthase 1−/− macrophages did not inhibit T cell proliferation. Furthermore, treatment of T cells with PGE2 recreated the effects seen with infected supernatant. From these data, we conclude that F. tularensis can alter host T cell responses by causing macrophages to produce PGE2. This study defines a previously unknown mechanism used by F. tularensis to modulate adaptive immunity.

Lipid Raft-Independent B Cell Receptor-Mediated Antigen Internalization and Intracellular Trafficking

The Ag-specific B cell receptor (BCR) expressed by B lymphocytes has two distinct functions upon interaction with cognate Ag: signal transduction (generation of intracellular second messenger molecules) and Ag internalization for subsequent processing and presentation. While it is known that plasma membrane domains, termed lipid rafts, are involved in BCR-mediated signal transduction, the precise role of plasma membrane lipid rafts in BCR-mediated Ag internalization and intracellular trafficking is presently unclear. Using a highly characterized model system, it was determined that while plasma membrane lipid rafts can be internalized by B lymphocytes, lipid rafts do not represent a major pathway for the rapid and efficient internalization of cell surface Ag-BCR complexes. Moreover, internalized plasma membrane lipid rafts are delivered to intracellular compartments distinct from those to which the bulk of internalized Ag-BCR complexes are delivered. These results demonstrate that B lymphocytes, like other cell types, possess at least two distinct endocytic pathways (i.e., clathrin-coated pits and plasma membrane lipid rafts) that deliver internalized ligands to distinct intracellular compartments. Furthermore, Ag-BCR complexes differentially access these two distinct internalization pathways.

Francisella tularensis Induces Ubiquitin-Dependent Major Histocompatibility Complex Class II Degradation in Activated Macrophages

ABSTRACT The intracellular bacterium Francisella tularensis survives and replicates within macrophages, ultimately killing the host cell. Resolution of infection requires the development of adaptive immunity through presentation of F. tularensis antigens to CD4+ and CD8+ T cells. We have previously established that F. tularensis induces macrophage prostaglandin E2 (PGE2) production, leading to skewed T-cell responses. PGE2 can also downregulate macrophage major histocompatibility complex (MHC) class II expression, suggesting that F. tularensis-elicited PGE2 may further alter T-cell responses via inhibition of class II expression. To test this hypothesis, gamma interferon (IFN-γ)-activated reporter macrophages were exposed to supernatants from F. tularensis-infected macrophages, and the class II levels were measured. Exposure of macrophages to infection supernatants results in essentially complete clearance of surface class II and CD86, compromising the macrophages ability to present antigens to CD4 T cells. Biochemical analysis revealed that infection supernatants elicit ubiquitin-dependent class II downregulation and degradation within intracellular acidic compartments. By comparison, exposure to PGE2 alone only leads to a minor decrease in macrophage class II expression, demonstrating that a factor distinct from PGE2 is eliciting the majority of class II degradation. However, production of this non-PGE2 factor is dependent on macrophage cyclooxygenase activity and is induced by PGE2. These results establish that F. tularensis induces the production of a PGE2-dependent factor that elicits MHC class II downregulation in IFN-γ-activated macrophages through ubiquitin-mediated delivery of class II to lysosomes, establishing another mechanism for the modulation of macrophage antigen presentation during F. tularensis infection.

Major Histocompatibility Complex (MHC) Class II-Peptide Complexes Arrive at the Plasma Membrane in Cholesterol-rich Microclusters

Background: Antigen-specific CD4 T cells are activated by small numbers of antigenic peptide-MHC class II (pMHC-II) complexes on dendritic cells (DCs). Results: Newly generated pMHC-II complexes are present in small clusters on the DC surface. Conclusion: pMHC-II clusters permit efficient T cell activation. Significance: The appearance of clustered pMHC-II reveals the organization of the T cell antigen receptor ligand on the DC surface. Dendritic cells (DCs) function by stimulating naive antigen-specific CD4 T cells to proliferate and secrete a variety of immunomodulatory factors. The ability to activate naive T cells comes from the capacity of DCs to internalize, degrade, and express peptide fragments of antigenic proteins on their surface bound to MHC class II molecules (MHC-II). Although DCs express tens of thousands of distinct MHC-II, very small amounts of specific peptide-MHC-II complexes are required to interact with and activate T cells. We now show that stimulatory MHC-II I-Ak-HEL(46–61) complexes that move from intracellular antigen-processing compartments to the plasma membrane are not randomly distributed on the DC surface. Confocal immunofluorescence microscopy and quantitative immunoelectron microscopy reveal that the majority of newly generated MHC-II I-Ak-HEL(46–61) complexes are expressed in sub-100-nm microclusters on the DC membrane. These microclusters are stabilized in cholesterol-containing microdomains, and cholesterol depletion inhibits the stability of these clusters as well as the ability of the DCs to function as antigen-presenting cells. These results demonstrate that specific cohorts of peptide-MHC-II complexes expressed on the DC surface are present in cholesterol-dependent microclusters and that cluster integrity is important for antigen-specific naive CD4 T cell activation by DCs.

The Pathway of Antigen Uptake and Processing Dictates MHC Class II-Mediated B Cell Survival and Activation

The influence of the pathway of Ag uptake and processing on MHC class II (CII)-mediated B cell function is unknown. In this study, we investigate in resting and activated (via the BCR or CD40) B cells the biological properties of CII-peptide complexes (CII-peptide) generated by either the BCR-mediated Ag processing (type I complex) or fluid phase Ag processing (type II complex). Compared with type I complex, ligation of type II complex by either specific Ab or the TCR in Ag-presenting assay results in significant decreases in B cell survival rate (50–100%) and expression levels of CII, CD86, and CD54. Loss of B cells following ligation of type II complex occurs in the presence of a comparatively good level of specific CD4+ T cell division, indicating that B cell loss is a late event following T cell stimulation. Comparative analysis of T and B cell conjugates after Ab ligation of type I or II complex reveals decreased efficiency of the latter in forming conjugates. Neither initial differential levels of CII and other studied surface markers, B cell type inherent differences, BCR signaling, T cell proliferation, nor initial density of CII-peptide complexes could explain the T cell-induced B cell loss. We propose that the context in which CII-peptide complexes are present in the membrane following BCR uptake and processing leads to B cell survival. Thus, appropriate targeting of Ag ensures generation of relevant immune responses.

Dynamics of MHC class II-activating signals in murine resting B cells.

MHC class II (MHC II) proteins are competent signaling molecules on APC. However, little is known about the mechanisms that control generation of their activating signals. Previous reports highlighted a number of factors that could affect the nature and outcome of MHC II signals, including the inability of MHC II ligation on resting vs activated murine B cells to induce mobilization of Ca2+. In the present study, we report that ligation of MHC II on resting murine B cells reproducibly induces mobilization of intracellular Ca2+ using both mAbs and cognate T cells as ligands. Mobilization of Ca2+ was independent of MHC II haplotype, isotype, or mouse genetic background. MHC II-mediated mobilization of Ca2+ is completely inhibited by inhibitors of src-like kinases and syk, and MHC II ligation increases overall tyrosine phosphorylation level. Moreover, MHC II ligation results in specific up-regulation of CD86. However, induction of these responses is dependent on the type of anti-MHC II Ab used, suggesting that epitope specificity and/or the nature of ligation is important. Moreover, we demonstrate that MHC II-derived signals are strictly regulated by the order and timing of BCR and CD40 signals, suggesting coordination of these signals preserves the integrity of early B cell priming events. Thus, the mode and the context of MHC II ligation influence generation of MHC II-derived activating signals in resting B cells. Based on these results, a new model that highlights the role of MHC II-activating signals in regulation of Ag presentation by B cells is proposed.

Functional and structural requirements for the internalization of distinct BCR-ligand complexes.

Antigen (Ag) binding to the BCR rapidly initiates two important events: a phosphorylation cascade that results in the production of secondary signaling intermediaries and the internalization of Ag‐BCR complexes. Previous studies using anti‐BCR antibodies (Ab) have suggested that BCR signaling is an essential requirement for BCR endocytosis and have further implicated lipid rafts as essential platforms for both BCR functions. However, published data from our laboratory indicate that lipid rafts and consequently raft‐mediated signaling are dispensable for BCR‐mediated internalization of Ag‐specific BCR. Therefore, we investigated the relationship between BCR signaling and endocytosis by defining the role of early kinase signaling in the BCR‐mediated internalization of a model Ag (haptenated protein). The results demonstrate that Src kinases and Syk‐mediated BCR signaling are not essential for BCR‐mediated Ag internalization. Moreover, by comparing Ag and Ab, it was determined that while both localize to clathrin‐coated pits, the internalization of Ab‐BCR complexes is more susceptible to inhibition of signaling and highly sensitive to disruption of lipid rafts and the actin cytoskeleton compared to Ag‐BCR complexes. Thus, these results demonstrate that the nature of the ligand ultimately determines the functional requirements and relative contribution of lipid rafts and other membrane structures to the internalization of BCR‐ligand complexes.

Francisella tularensis Elicits IL-10 via a PGE2-Inducible Factor, to Drive Macrophage MARCH1 Expression and Class II Down-Regulation

Francisella tularensis is a bacterial pathogen that uses host-derived PGE2 to subvert the hosts adaptive immune responses in multiple ways. Francisella-induced PGE2 acts directly on CD4 T cells to blunt production of IFN-γ. Francisella-induced PGE2 can also elicit production of a >10 kDa soluble host factor termed FTMØSN (F. tularensis macrophage supernatant), which acts on IFN-γ pre-activated MØ to down-regulate MHC class II expression via a ubiquitin-dependent mechanism, blocking antigen presentation to CD4 T cells. Here, we report that FTMØSN-induced down-regulation of MØ class II is the result of the induction of MARCH1, and that MØ expressing MARCH1 “resistant” class II molecules are resistant to FTMØSN-induced class II down-regulation. Since PGE2 can induce IL-10 production and IL-10 is the only reported cytokine able to induce MARCH1 expression in monocytes and dendritic cells, these findings suggested that IL-10 is the active factor in FTMØSN. However, use of IL-10 knockout MØ established that IL-10 is not the active factor in FTMØSN, but rather that Francisella-elicited PGE2 drives production of a >10 kDa host factor distinct from IL-10. This factor then drives MØ IL-10 production to induce MARCH1 expression and the resultant class II down-regulation. Since many human pathogens such as Salmonella typhi, Mycobacterium tuberculosis and Legionella pneumophila also induce production of host PGE2, these results suggest that a yet-to-be-identified PGE2-inducible host factor capable of inducing IL-10 is central to the immune evasion mechanisms of multiple important human pathogens.

The Syk-binding Ubiquitin Ligase c-Cbl Mediates Signaling-dependent B Cell Receptor Ubiquitination and B Cell Receptor-mediated Antigen Processing and Presentation

Background: c-Cbl associates with various signaling molecules to regulate diverse signaling networks via ubiquitination of receptors and/or protein tyrosine kinases. Results: c-Cbl drives ubiquitin-dependent Ag·BCR trafficking to MIIC and BCR-mediated antigen processing and presentation. Conclusion: c-Cbl directs BCR-mediated antigen processing and presentation. Significance: c-Cbl coordinates antigen-induced BCR signaling and rapid BCR-mediated antigen processing and presentation to drive a strong humoral immune response. B cell receptor (BCR)-mediated antigen (Ag) processing and presentation lead to B cell-T cell interactions, which support affinity maturation and immunoglobulin class switching. These interactions are supported by generation of peptide-MHC class II complexes in multivesicular body-like MIIC compartments of B cells. Previous studies have shown that trafficking of Ag·BCR complexes to MVB-like MIIC occurs via an ubiquitin-dependent pathway and that ubiquitination of Ag·BCR complexes occurs by an Src family kinase signaling-dependent mechanism that is restricted to lipid raft-resident Ag·BCR complexes. This study establishes that downstream Syk-dependent BCR signaling is also required for BCR ubiquitination and BCR-mediated antigen processing and presentation. Knockdown studies reveal that of the two known Syk-binding E3 ubiquitin ligases c-Cbl and Cbl-b, only c-Cbl appears to have a central role in BCR ubiquitination, trafficking to MIIC, and ubiquitin-dependent BCR-mediated antigen processing and presentation. These results establish the novel role for Syk signaling and the Syk-binding ubiquitin ligase c-Cbl in the BCR-mediated processing and presentation of cognate antigen and define one mechanism by which antigen-induced BCR ubiquitination is modulated to impact the initiation and maturation of the humoral immune response.

Differential Transmembrane Domain GXXXG Motif Pairing Impacts Major Histocompatibility Complex (MHC) Class II Structure

Background: Major histocompatibility complex class II molecules are structurally and functionally heterogeneous. Results: Combined mutagenesis and structural studies establish a role for pairing between conserved transmembrane (TM) GXXXG dimerization motifs in determining class II conformation. Conclusion: Differential pairing of highly conserved TM domain dimerization motifs contributes to class II structure and function. Significance: Global conformation contributes to the function of peptide-class II complexes. Major histocompatibility complex (MHC) class II molecules exhibit conformational heterogeneity, which influences their ability to stimulate CD4 T cells and drive immune responses. Previous studies suggest a role for the transmembrane domain of the class II αβ heterodimer in determining molecular structure and function. Our previous studies identified an MHC class II conformer that is marked by the Ia.2 epitope. These Ia.2+ class II conformers are lipid raft-associated and able to drive both tyrosine kinase signaling and efficient antigen presentation to CD4 T cells. Here, we establish that the Ia.2+ I-Ak conformer is formed early in the class II biosynthetic pathway and that differential pairing of highly conserved transmembrane domain GXXXG dimerization motifs is responsible for formation of Ia.2+ versus Ia.2− I-Ak class II conformers and controlling lipid raft partitioning. These findings provide a molecular explanation for the formation of two distinct MHC class II conformers that differ in their inherent ability to signal and drive robust T cell activation, providing new insight into the role of MHC class II in regulating antigen-presenting cell-T cell interactions critical to the initiation and control of multiple aspects of the immune response.