Physical Characterization and Innate Immunogenicity of Aggregated Intravenous Immunoglobulin (IGIV) in an In Vitro Cell-Based Model.

PURPOSE: To investigate in vitro the innate immune response to accelerated stress-induced aggregates of intravenous immunoglobulin (IGIV) using a well-defined human cell-line model, and to correlate the innate response to physical properties of the aggregates. METHODS: IGIV aggregates were prepared by applying various accelerated stress methods, and particle size, count and structure were characterized. Immune cell activation as tracked by inflammatory cytokines released in response to aggregates was evaluated in vitro using peripheral blood mononuclear cells (PBMC), primary monocytes and immortalized human monocyte-like cell lines. RESULTS: IGIV aggregates produced by mechanical stress induced higher cytokine release by PBMC and primary monocytes than aggregates formed by other stresses. Results with the monocytic cell line THP-1 paralleled trends in PBMC and primary monocytes. Effects were dose-dependent, enhanced by complement opsonization, and partially inhibited by blocking toll-like receptors (TLR2 and TLR4) and to a lesser extent by blocking Fc gamma receptors (FcγRs). CONCLUSIONS: Stress-induced IGIV aggregates stimulate a dose-dependent cytokine response in human monocytes and THP-1 cells, mediated in part by TLRs, FcγRs and complement opsonization. THP-1 cells resemble primary monocytes in many respects with regard to tracking the innate response to IgG aggregates. Accordingly, the measurement of inflammatory cytokines released by THP-1 cells provides a readily accessible assay system to screen for the potential innate immunogenicity of IgG aggregates. The results also highlight the role of aggregate structure in interacting with the different receptors mediating innate immunity.

Cytoplasmic processing is a prerequisite for presentation of an endogenous antigen by major histocompatibility complex class II proteins.

Biochemical and functional studies have demonstrated major histocompatibility complex (MHC) class II-restricted presentation of select epitopes derived from cytoplasmic antigens, with few insights into the processing reactions necessary for this alternate pathway. Efficient presentation of an immunodominant epitope derived from glutamate decarboxylase (GAD) was observed regardless of whether this antigen was delivered exogenously or via a cytoplasmic route into human histocompatibility leukocyte antigen class II-DR4(+) antigen-presenting cells. Presentation of exogenous as well as cytoplasmic GAD required the intersection of GAD peptides and newly synthesized class II proteins. By contrast, proteolytic processing of this antigen was highly dependent upon the route of antigen delivery. Exogenous GAD followed the classical pathway for antigen processing, with an absolute requirement for endosomal/lysosomal acidification as well as cysteine and aspartyl proteases resident within these organelles. Presentation of endogenous GAD was dependent upon the action of cytoplasmic proteases, including the proteasome and calpain. Thus, translocation of processed antigen from the cytoplasm into membrane organelles is necessary for class II-restricted presentation via this alternate pathway. Further trimming of these peptides after translocation was mediated by acidic proteases within endosomes/lysosomes, possibly after or before class II antigen binding. These studies suggest that processing of exogenous and cytoplasmic proteins occurs through divergent but overlapping pathways. Furthermore, two cytoplasmic proteases, the proteasome and calpain, appear to play important roles in MHC class II-restricted antigen presentation.

Coordinate defects in human histocompatibility leukocyte antigen class II expression and antigen presentation in bare lymphocyte syndrome.

The human immunodeficiency, type II bare lymphocyte syndrome (BLS), has been attributed to a defect in the transcription of class II histocompatibility genes. Immunocompetence, as assessed by functional exogenous antigen presentation, was not restored in immortalized B cells, derived from a BLS patient, after transfection with HLA-DR class II structural genes. Incubation of protein antigens, as well as infectious virus, with DR-transfected BLS cells failed to induce activation of antigen-specific helper T lymphocytes. Peptide antigens were presented by class II molecules displayed on BLS cells, although the conformation of these class II proteins was altered as indicated by epitope mapping. This defect in antigen presentation was independent of the specific class II DR allele transfected into BLS cells. Genetic complementation analysis has been used with BLS cells to demonstrate that the defect in class II gene transcription is linked to the absence of a trans-acting factor. Similarly, functional class II dimers were restored after in vitro fusion of cells derived from two distinct BLS complementation groups, implying that specific transcriptional control elements are shared by a gene critical for antigen presentation and genes encoding HLA class II antigens. Thus, two important functionally linked pathways of class II molecules, structural gene expression and antigen presentation, share a common regulatory pathway defective in BLS.

Invariant chain trimers are sequestered in the rough endoplasmic reticulum in the absence of association with HLA class II antigens.

HLA class II antigens are heterodimeric cell surface glycoproteins that interact with antigenic peptides to form complexes recognizable by CD4-positive T cells. During their biosynthesis, class II antigens are retained in a post-Golgi compartment in association with the invariant chain, which dissociates before class II cell surface expression. To address whether the invariant chain mediates this post-Golgi retention, its transport and assembly were examined in cells that do not express HLA class II antigens. Pulse-chase analysis and endoglycosidase digestions showed that very little invariant chain proceeded as far as the trans-Golgi in class II-negative cell lines. Immunofluorescence studies suggested that in these cells the invariant chain is sequestered in the RER. Gel filtration and cross-linking data showed that RER-localized invariant chain is present as trimers or aggregated trimers. Multimerization is mediated by lumenal interactions; a proteolytic fragment of the invariant chain corresponding to the lumenal domain remained trimeric as determined by cross-linking analysis. Similar transport and structural characteristics were observed for a pool of excess invariant chain in class II-positive cells, suggesting that an excess of invariant chain in the ER may be important for class II antigen function. These results have important implications for the transport of cellular proteins in general and for the role of the invariant chain in class II antigen biosynthesis.

Deficient antigen-presenting cell function in multiple genetic complementation groups of type II bare lymphocyte syndrome.

The absence of HLA class II gene expression in type II bare lymphocyte syndrome (BLS) results from defective transcriptional activation of class II histocompatibility genes. Genetic studies have revealed that distinct defects in multiple trans-acting factors result in the immunodeficient BLS phenotype. We studied antigen-presenting cell (APC) function in DR-transfected BLS cells derived from multiple complementation groups. Each BLS cell line displayed the same defective APC phenotype: an inability to mediate class II-restricted presentation of exogenous protein antigens, and structurally altered class II alpha beta dimers. Expression of the HLA class II-like genes DMA and DMB, previously implicated in antigen presentation, was reduced or absent in the BLS cells. Fusion of BLS cells with cell line 721.174, which has a genomic deletion of HLA class II genes, coordinately restores class II structural gene and DM gene expression and a wild-type APC phenotype. Thus each of the molecular defects that silences class II structural gene transcription also results in a defective APC phenotype, providing strong evidence for coregulation of these two functionally linked pathways.

Intestinal epithelial cells use two distinct pathways for HLA class II antigen processing.

Intestinal epithelial cells express a low level of HLA class II molecules constitutively, with elevated levels seen in the setting of mucosal inflammation including inflammatory bowel disease. The ability of intestinal epithelial cells to act as antigen presenting cells for alphabeta CD4(+) T lymphocytes was examined through a molecular analysis of the HLA class II antigen processing pathway. We have shown that intestinal epithelial cells contain abundant constitutive levels of the cathepsin proteases proven to function in HLA class II mediated antigen presentation. Activation of these cells by gamma-IFN induced the expression of invariant chain and HLA-DM alphabeta, thus facilitating the formation of compact, SDS-stable HLA- DR alphabeta heterodimers. Using HLA-DR-restricted T cells and retroviral mediated gene transfer of HLA-DR alleles into the intestinal epithelial cell lines HT-29 and T84, we demonstrated efficient antigen processing and presentation to CD4(+) T lymphocytes in the presence of the proinflammatory cytokine gamma-IFN. The class II processing pathway and presentation in the presence of gamma-IFN was indistinguishable from that observed with a conventional antigen presenting cell. Antigen processing also occurred in intestinal epithelial cells in the absence of gamma-IFN, and in contrast to that seen after stimulation with gamma-IFN, required high concentration of antigen and was not inhibited by the protease inhibitor leupeptin. These data suggest the use of two distinct pathways of HLA class II antigen processing in enterocytes with differential immunomodulatory properties in the presence or absence of mucosal inflammation.

Antigen-presenting cells and the selection of immunodominant epitopes.

Cellular immune responses are directed against a narrow set of immunodominant peptides derived from complex antigens. By contrast, epitopes that are hidden or infrequent targets of immune responses have been termed cryptic or subdominant. Although the identification of immunodominant epitopes is important for vaccine development, understanding immunological reactivity to cryptic or subdominant epitopes may hold clues to autoimmunity. We have examined the role of antigen-presenting (APC) cells in the selection of class Ii-restricted epitopes for display to T lymphocytes. The formation and MHC-restricted presentation of distinct antigenic epitopes is directly dependent upon processing and ligand binding reactions within APC. A novel MHC heterodimer, HLA-DM, facilitates the binding and presentation of peptides by class II DR, DP, and DQ molecules. We have demonstrated that some epitopes derived from endogenous antigens bind class II proteins independent of DM, whereas the presentation of other endogenous peptides is greatly influenced by DM expression. Targeting of exogenous antigens into specialized processing compartments within APC appears to overcome the requirement for DM in antigen presentation. These studies suggest HLA-DM may play a role in epitope selection and immunodominance.

Biosynthesis and processing of class II histocompatibility antigens.

The class II major histocompatibility antigens at the cell surface exist as heterodimers of alpha and beta subunits. During biosynthesis, these subunits are associated with a third chain, the invariant (I) or I chain. Association with the I chain occurs early in biosynthesis in the rough endoplasmic reticulum and persists during transport through the Golgi apparatus. One of the two alpha subunit N-linked oligosaccharides and the single beta subunit N-linked oligosaccharide are converted to the complex form during Golgi transit. In the human system, both I chain N-linked oligosaccharides can also be processed to the complex form, and at least two O-linked oligosaccharides can be added to the I chain. At some point during transit to the cell surface, class II antigens associate with a proteoglycan bearing chondroitin sulfate side chains. Complexes containing alpha, beta and I chain subunits and the associated proteoglycan accumulate in human B-cell lines treated with the ionophore monensin, an inhibitor of Golgi transport, suggesting that this may be a biosynthetic intermediate in class II antigen transport and assembly. Prior to cell surface expression of class II antigens, the exocytic pathway which they follow intersects the endocytic route, followed by certain ligands internalized by receptor-mediated endocytosis. The I chain appears to dissociate from mature class II alpha, beta dimers prior to their cell surface expression but following the intersection of the exocytic and endocytic pathways.

New insights in antigen processing and epitope selection: development of novel immunotherapeutic strategies for cancer, autoimmunity and infectious diseases.

Current evidence suggests that MHC class II-restricted CD4+ T-cells play a crucial role in orchestrating host immune responses against cancer as well as autoimmune and infectious diseases. Antigens must be processed within endosomal and lysosomal compartments of antigen presenting cells (APC) before binding to MHC class II molecules for display to T-cells. Only a limited number of processed peptides termed immunodominant are selected for display by MHC class II molecules and prove capable of inducing strong T-cell responses. Thus processing reactions within APC are of central importance for the development of effective vaccines as they modulate the number of peptide: class II complexes by enhancing or disrupting epitope formation and display. Studies suggest that there are substantial gaps in our knowledge of how antigen processing and presentation by APC regulates epitope selection and immunodominance in disease situations. Here we describe new insights in antigen processing and epitope selection with relevance to immunotherapeutic strategies for cancer, autoimmunity and infectious diseases.

Regulation of mannose receptor synthesis and turnover in mouse J774 macrophages.

The mannose receptor, present on the plasma membrane of macrophages, promotes the internalization of glycoproteins and glycoconjugates via both endocytic and phagocytic pathways. The expression of this receptor is tightly modulated during monocyte/Mphi differentiation and cellular activation. We isolated clonal populations from murine J774 macrophage tumor cells, which differ in their surface expression of functional mannose receptors. To examine the potential mechanisms regulating receptor function in these cell lines, the interaction of receptor with ligand as well as receptor synthesis and degradation was analyzed. J774 clones with both high and low levels of mannose receptor activity were found to synthesize significant amounts of receptor protein, suggesting that the protein may be regulated at the level of synthesis and degradation. In J774 clones expressing very low receptor activity and protein, the half-life of mannose receptor molecules was substantially decreased. The evolution of multiple mechanisms modulating mannose receptor function may be critical in fine-tuning the role of this receptor in antigen processing and in scavenger and host defense functions.

Detection of biotinylated cell surface receptors and MHC molecules in a capture ELISA: a rapid assay to measure endocytosis.

Cell surface receptors and antigens, such as TfR and MHC molecules, are endocytosed and subsequently redisplayed on the plasma membrane. The internalization and recycling of MHC molecules is thought to play an important role in antigen presentation, but studying this process has been hindered due to the lack of a rapid and easily quantitated assay. The combination of a cleavable biotin reagent to label surface molecules and a capture ELISA to detect these molecules of interest, allows for the quantitation of their cell surface expression, endocytosis and recycling. The endocytosis of TfR and MHC II molecules was readily quantitated in B cell lines using this procedure with results nearly identical to previously published data using more laborious radioactive methods. Evidence for the recycling of class II antigens and TfR back to the plasma membrane was obtained by monitoring the exit of these molecules from endosomes. Exposing cells to hypertonic media blocks clathrin-dependent endocytosis and was found to inhibit the internalization of MHC class II proteins on B cells. This flexible assay to capture and quantitate the cell surface expression and endocytosis of MHC molecules and other surface antigens offers a sensitive and non-radioactive alternative to study the intracellular trafficking of diverse membrane proteins.

Modulation of peptide-dependent allospecific epitopes on HLA-DR4 molecules by HLA-DM.

Peptide binding to HLA-DR molecules in intracellular compartments is facilitated by HLA-DM molecules, present in most types of antigen-presenting cells. Allorecognition of DR specificities represents a form of T cell recognition of the MHC-peptide complex which in some cases is influenced by peptide binding. DRA and DRB*0401 (Dw4) genes were introduced into different cell types including DM-negative and DM-restored mutant cells to analyze recognition of DR4 subtypes by alloreactive T cell clones and Dw4-specific monoclonal antibodies. Distinct patterns of T cell recognition were identified: (i) deficient response to Dw4 molecules in the absence of DM expression in which T cell responses were restored by transfecting DM into the Dw4-expressing cells; and (ii) equivalent recognition of Dw4 on DM- and DM+ cells. Using several mAb to Dw4 molecules, a similar distinction was observed: a shared epitope on Dw4 and Dw14 molecules was partially DM-independent while a Dw4-specific epitope was DM-dependent and cell type-specific. Thus, a subset of both T cell and mAb allodeterminants are influenced by a DM-dependent interaction of MHC molecules with peptides, while the formation of DM-independent allodeterminants may represent direct MHC epitope recognition by the T cell receptor or an alternative peptide loading mechanism distinct from the HLA-DM pathways.

Purification and characterization of the D-mannose receptor from J774 mouse macrophage cells.

Macrophages display on their cell surface a D-mannose-specific receptor which facilitates the scavenging of certain pathogens and deleterious macromolecules from the extracellular fluid as part of the host defense mechanism. The mouse D-mannose receptor was purified from J774 E macrophages and an antiserum was generated against the receptor protein. In mouse macrophages, the newly synthesized receptor has an Mr of 157,000 Da and rapidly matures to a protein with an Mr of 172,000 Da. Both forms of the receptor protein are tightly associated with cell membranes. The receptor is found in a number of mouse macrophage cell types but is not present in mouse fibroblasts. An assay was developed to characterize D-mannose receptor-ligand binding based on immunoprecipitation of the detergent-solubilized receptor protein. The dissociation constant, determined for receptor and the neoglycoprotein D-mannose-BSA, was 1.67nM. Receptor-ligand binding was calcium and pH dependent. Monosaccharides, such as D-mannose and L-fucose, partially inhibited receptor binding to the ligand D-mannose-BSA.

Anti-type V collagen lymphocytes that express IL-17 and IL-23 induce rejection pathology in fresh and well-healed lung transplants.

Immunity to collagen V [col(V)] contributes to lung 'rejection.' We hypothesized that ischemia reperfusion injury (IRI) associated with lung transplantation unmasks antigenic col(V) such that fresh and well-healed lung grafts have differential susceptibility to anti-col(V)-mediated injury; and expression of the autoimmune cytokines, IL-17 and IL-23, are associated with this process. Adoptive transfer of col(V)-reactive lymphocytes to WKY rats induced grade 2 rejection in fresh isografts, but induced worse pathology (grade 3) when transferred to isograft recipients 30 days post-transplantation. Immunhistochemistry detected col(V) in fresh and well-healed isografts but not native lungs. Hen egg lysozyme-reactive lymphocytes (HEL, control) did not induce lung disease in any group. Col(V), but not HEL, immunization induced transcripts for IL-17 and IL-23 (p19) in the cells utilized for adoptive transfer. Transcripts for IL-17 were upregulated in fresh, but not well-healed isografts after transfer of col(V)-reactive cells. These data show that IRI predisposes to anti-col(V)-mediated pathology; col(V)-reactive lymphocytes express IL-17 and IL-23; and anti-col(V)-mediated lung disease is associated with local expression of IL-17. Finally, because of similar histologic patterns, the pathology of clinical rejection may reflect the activity of autoimmunity to col(V) and/or alloimmunity.

Role for intracellular proteases in the processing and transport of class II HLA antigens.

Human B-lymphoblastoid cell lines (B-LCL) incubated with the protease inhibitor leupeptin accumulate complexes of class II HLA antigens with a series of Mr 21,000-23,000 basic proteins termed leupeptin-induced proteins (LIP). The appearance of class II antigen-associated LIP coincides with the disappearance of class II antigen-associated invariant (I) chain. Glycopeptides generated by in vitro proteolysis of LIP and I chain using Staphylococcus aureus V8 protease are identical as determined by electrophoresis in sodium dodecyl sulfate. These results suggest that LIP is a proteolytic product derived from the I chain and are consistent with the view that further in vivo proteolysis of LIP by a leupeptin-sensitive enzyme normally facilitates its release from class II antigens. Incubation of B-LCL with monensin, which traps class II antigens and associated I chain in the Golgi apparatus, or chloroquine, which neutralizes intracellular acidic compartments and inhibits I-chain dissociation, blocks the leupeptin-induced appearance of LIP. Treatment of LIP with endoglycosidases F and H shows that both of its N-linked oligosaccharides are in the complex form, indicating that proteolysis of class II antigen-associated I chain to generate LIP occurs in a late-Golgi or post-Golgi compartment. The compartment in which these proteolytic events occur may be identical to the site in macrophages and B lymphocytes where foreign antigens are processed and interact with class II HLA molecules.