Presentation of Listeria monocytogenes antigens by major histocompatibility complex class I molecules to CD8 cytotoxic T lymphocytes independent of listeriolysin secretion and virulence

Virulence and intracellular persistence of Listeria monocytogenes markedly depend on secretion of listeriolysin (Hly), which promotes invasion of the pathogen from the endosome into the cytosol. Recent studies have provided compelling evidence that Hly also facilitates recognition of listerial antigens, in association with major histocompatibility complex (MHC) class I molecules, by CD8 T lymphocytes. Data presented here confirm that the Hly-deficient strains, the prfA^? mutant L. monocytogenes SLCC53 and the transposon mutants L. monocytogenes M3 and M20 are avirulent for mice, and unable to replicate inside bone marrow-derived macrophages (BMMΦ). Furthermore, BMMΦ infected with M3, M20 or SLCC53 were as efficiently lysed as BMMΦ infected with the Hly-positive wild-type strain EGD by MHC class I-dependent CD8 cytotoxic T lymphocytes. Using the highly sensitive polymerase chain reaction method, hly mRNA was detectable in BMMΦ infected with L. monocytogenes EGD or SLCC53, but totally absent in M3-infected BMMΦ. In the case of M20, an excision of the transposon occurred, but the excision was not precise and the hly gene was approximately 400 base pairs shorter. These findings argue against a unique role for Hly in MHC class I presentation of listerial antigens, although Hly appears central to virulence and intracellular replication. Thus, virulence of L. monocytogenes is dissociable from MHC class I presentation of listerial antigens.     

Excessive degradation of intracellular protein in macrophages prevents presentation in the context of major histocompatibility complex class II molecules

The endogenous major histocompatibility complex (MHC) class II presentation pathway allows biosynthesized, intracellular antigens access for presentation to MHC class II-restricted T cells. This pathway has been well documented in B cells and fibroblasts, but may not be universally available in all antigen-presenting cell types. This study compares the ability of different antigen-presenting cells, expressing endogenous C5 protein (fifth component of mouse complement) as a result of transfection, to present their biosynthesized C5 to MHC class II-restricted T cells. B cells and fibroblasts expressing C5 were able to present several epitopes of this protein with MHC class II molecules, whereas macrophages were unable to do so, but readily presented C5 from an extracellular source. However, macrophage presentation of endogenous C5 could be achieved when they were treated with low doses of the lysosomotropic agent ammonium chloride. In the presence of an inhibitor of autophagy, presentation of endogenous C5 was abrogated, indicating that biosynthesized C5 is shuttled into lysosomal compartments for degradation before making contact with MHC class II molecules. Taken together, this suggests that proteolytic activity in lysosomes of macrophages may be excessive, compared with fibroblasts and B cells, and destroys epitopes of the C5 protein before they can gain access to MHC class II molecules. Thus, there are inherent differences in presentation pathways between antigen-presenting cell types; this could reflect their specialized functions within the immune system with macrophages focussing preferentially on internalization, degradation, and presentation of extracellular material.     

Epitope selection in major histocompatibility complex class I-mediated pathway is affected by the intracellular localization of an antigen

We analyzed the mode of antigen presentation of an endogenous antigen localized in the cytoplasm or in the mitochondria. Pseudomonas aeruginosa PAO leucine-, isoleucine-, valine-binding protein (LIVAT-BP) encoded by the braC gene was used as a model antigen. Using mouse BALB/3T3 cells, we established two LIVAT-BP transfectants by transfection of a plasmid harboring the intact braC or braC gene fused with the mitochondrial transport signal derived from the yeast COXIV gene. One of the resulting transfectants, BC-15, expressed LIVAT-BP in the cytoplasm, while YZ-710 cells expressed LIVAT-BP in the mitochondria. The splenic effector cells derived from BALB/c mice primed with BC-15 cells exhibited cytotoxic T lymphocyte (CTL) activity against BC-15 cells, but not against YZ-710 cells, whereas splenic effector cells primed with YZ-710 cells exhibited CTL activity against YZ-710 cells, but not against BC-15 cells. Neither group of splenic effector cells showed CTL activity against parental BALB/3T3 cells. These CTL belonged to the CD8⁺ αβ T cell subset. Furthermore, we observed that the CTL activity against BC-15 cells or YZ-710 cells was blocked with anti-H2-K^d mAb, but not with anti-H2-D^d or H2-L^d mAb. The CTL against BC-15 or YZ-710 cells could kill parental BALB/3T3 cells in the presence of peptides produced by alkali lysis of the LIVAT-BP. suggesting that these CTL indeed recognized the peptide(s) derived from LIVAT-BP. We determined that the epitope for the CTL against BC-15 cells was QYGEGIATEV, corresponding to residues 162–171, and that the epitope recognized by the CTL against YZ-710 cells was GYKLIFRTI, corresponding to residues 123–131 of LIVAT-BP, respectively. Thus, we show here that epitope selection for MHC class I expression is affected by the intracellular localization of the antigenic protein.     

Constitutive macropinocytosis allows TAP-dependent major histocompatibility compex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells

Dendritic cells expanded from mouse bone marrow (BMDC) with granulocyte/macrophage-colony-stimulating factor have potent T cell-stimulatory properties both in vitro and in vivo. This has been well documented for major histocompatibility complex (MHC) class II-restricted responses, and more recently using peptide-loaded and protein-pulsed DC for CD8 responses following adoptive transfer in mice. An unresolved question concerns the capacity of BMDC to present exogenous antigen on MHC class I molecules, an unconventional mode of MHC class I loading for which there is now considerable evidence, particularly in macrophages. Here, we show that BMDC exhibit high levels of macropinocytosis driven by constitutive membrane ruffling activity. Up to one-third of actively ruffling and macropinocytosing BMDC transferred pinocytosed horseradish peroxidase into the cytosol following a 15-min pulse, suggesting that they might be capable of presenting exogenous soluble antigen on MHC class I molecules. We show that BMDC presented exogenous ovalbumin to a T cell hybridoma more effectively, more rapidly, and at lower exogenous antigen concentrations than BM macrophages on a cell-for-cell basis. Presentation was TAP dependent, brefeldin A sensitive, and blocked by inhibitors of proteasomal processing, demonstrating use of the classical MHC class I pathway. Although effective presentation of exogenous antigen by BMDC occurred in the absence of agents which stimulate macropinocytosis, treatment with phorbol myristate acetate (PMA) enhanced both pinocytosis and MHC class I presentation by BMDC. Finally, PMA-stimulated BMDC exposed to exogenous ovalbumin in vitro were able to prime an antigen-specific cytotoxic T lymphocyte response following adoptive transfer in vivo.     

Peptide transporter-independent,stress protein-mediated endosomal processing of endogenous protein antigens for major histocompatibility complex class I presentation

The peptide transporter-defective cell line RMA-S expressing the wild-type simian virus 40 large T antigen (wtT-Ag) from a transfected gene did not present two well-defined, H-2 class I (D^b)-restricted epitopes of T-Ag to cytotoxic T lymphocytes (CTL). Hence, “endogenous” processing and presentation of the wtT-Ag depended on a functional peptide transporter heterodimer. In contrast, both T-Ag epitopes were efficiently presented to CTL by transfected RMA-S cells expressing a truncated, cytoplasmic T-Ag variant (cT-Ag) or a karyophilic, amino-terminal 272-amino acid T-Ag fragment. Transporter-independent “endogenous” processing of mutant T-Ag molecules correlated with their association with the constitutively expressed heat shock protein 73 (hsp 73). Class I-restricted presentation of both epitopes processed from these hsp73-associated protein antigens was sensitive to NH₄Cl and chloroquine. These data indicate that selected intracellular proteins access an alternative, hsp73-mediated pathway for class I-restricted presentation that operates independent of peptide transporters in an endosomal compartment.     

Presentation of exogenous antigens by macrophages: analysis of major histocompatibility complex class I and II presentation and regulation by cytokines

There is an antigen presenting cell (APC) in the lymphoid organs capable of presenting exogenous antigen (Ag) with major histocompatibility complex (MHC) class I molecules. This study was initiated to isolate clones of these APC to definitively establish their phenotype and to further study their properties. Murine bone marrow macrophages (BM MΦ) were immortalized by overexpressing myc and raf oncogenes. Five BM MΦ cell lines were generated that are phagocytic and expressed at their surface MΦ differentiation Ag. All five cell lines processed and presented exogenous ovalbumin (OVA) with MHC class I molecules. They all presented OVA-linked to a phagocytic substrate 10²–10⁴-fold more efficiently than soluble Ag. Clonal isolates of two of the MΦ cell lines had an identical phenotype and functional properties as the uncloned lines. These results definitively establish that MΦ are APC with the capacity of presenting exogenous Ag with MHC class I molecules. Interferon (IFN)-γ interleukin-4, granulocyte-macrophage colony stimulating factor and lipopolysaccharide either alone or in combination induced little or no augmentation and in some cases decreased presentation of exogenous OVA with MHC class I. In contrast, all of MΦ activating factors increased MHC class I expression. Moreover, IFN-γ increased the presentation of cytosolic OVA, demonstrating differences between the presentation of cytosolic Ag versus exogenous Ag with MHC class I. Finally, some lines constitutively processed and presented exogenous OVA with MHC class II while others only presented after stimulation with IFN-γ. These results demonstrate that the pathways involved in the presentation of exogenous Ag with MHC class I and class II are independently regulated and that a cloned cell is capable of presenting exogenous Ag through both pathways.     

Epitope cleavage by Leishmania endopeptidase(s) limits the efficiency of the exogenous pathway of major histocompatibility complex class I-associated antigen presentation

The activation of CD8⁺ T cell responses is commonplace during infection with a number of nonviral pathogens. Consequently, there has been much interest in the pathways of presentation of such exogenous antigens for major histocompatibility complex class I-restricted recognition. We had previously shown that Leishmania promastigotes transfected with the ovalbumin (OVA) gene could efficiently target OVA to the parasitophorous vacuole (PV), with subsequent recognition by class II-restricted T cells. We now report the results of studies aimed at evaluating the PV as a route of entry into the exogenous class I pathway. Bone marrow-derived macrophages can present soluble OVA (albeit at high concentrations) to the OVA_257–264-specific T cell hybridoma 13.13. In contrast, infection with OVA-transfected Leishmania promastigotes failed to result in the stimulation of this hybridoma. This appeared unrelated to variables such as antigen concentration, parasite survival, and macrophage activation status. These results prompted an analysis of the effects of promastigotes on class I peptide binding using RMA-S cells and OVA_257–264. Our data indicate that the major surface protease of Leishmania, gp63, inhibits this interaction by virtue of its endopeptidase activity against the OVA_257–264 peptide. The data suggest that this activity, if maintained within the PV, would result in loss of the OVA_257–264 epitope. Although we can therefore draw no conclusions from these studies regarding the efficiency of the PV as a site of entry of antigen into the exogenous class I pathway, we have identified a further means by which parasites may manipulate the immune repertoire of their host.     

Trypanosoma cruzi infection does not impair major histocompatibility complex class I presentation of antigen to cytotoxic T lymphocytes

Trypanosoma cruzi (T. cruzi), the etiological agent of Chagas' disease, lives free within the cytoplasm of infected host cells. This intracellular niche suggests that parasite antigens may be processed and presented on major histocompatibility complex (MHC) class I molecules for recognition by CD8⁺ T cells. However, the parasite persists indefinitely in the mammalian host, indicating its success at evading immune clearance. It has been shown that T. cruzi interferes with processing and presentation of antigenic peptides in the MHC class II pathway. This investigation sought to determine whether interference in MHC class I processing and presentation occurs with T. cruzi infection. Surface expression of MHC class I molecules was found to be unaffected or up-regulated by T. cruzi infection in vitro. A model system employing a β-galactosidase (β-gal)-specific murine cytotoxic T lymphocyte (CTL) line (0805B) showed: (i) in vitro infection of mouse peritoneal macrophages or J774 cells with T. cruzi did not inhibit MHC class I presentation of exogenous peptide (a nine-amino acid epitope of β-gal) to the CTL line, (ii) in vitro infection of a β-gal-expressing 3T3 cell line (LZEJ) with T. cruzi did not inhibit MHC class I presentation of the endogenous protein to the CTL line and (iii) mouse renal adenocarcinoma cells infected with T. cruzi and subsequently infected with adenovirus expressing β-gal were able to present antigen to the β-gal-specific CTL line. These findings indicate that the failure of the immune response to clear T. cruzi does not result from global interference by the parasite with MHC class I processing and presentation. Parasites engineered to express β-gal were unable to sensitize infected antigen-presenting cells in vitro to lysis by the CTL 0805B line. This was probably due to the intracellular localization of the β-gal within the parasite and its inaccessibility to the host cell cytoplasm.     

Targeting major histocompatibility complex class II molecules to the cell surface by invariant chain allows antigen presentation upon recycling

We studied the functional consequences of targeting class II molecules to either the cell surface or to endocytic structures by expressing HLA-DR1 in human kidney cells in the presence or absence of different forms of the invariant chain (Ii). Transfectants expressing class II molecules in the absence of Ii present influenza virus efficiently and co-expression of full length Ii does not further increase antigen presentation. Chimeric Ii containing the cytoplasmic domain of the transferrin receptor (Tfr-Ii) delivers class II molecules associated with Tfr-Ii to endosomal compartments, but this does not result in efficient antigen presentation. When class II molecules are targeted to the cell surface by Ii lacking either 15 (Δ15Ii) or 23 (Δ23Ii) amino acids from the cytoplasmic domain, a fraction of free class II molecules is also observed. Whereas Δ15Ii did not affect antigen presentation by class II molecules, Δ23Ii inhibited, but did not abrogate, the response. We show that class II molecules expressed in the presence of Δ23Ii can be internalized, followed by degradation of Δ23Ii and return of free class II αβ heterodimers to the cell surface. A fraction of the resulting free class II molecules is sodium dodecyl sulfate stable, indicating that internalization and reappearance of class II molecules at the cell surface can be an alternative route for antigen presentation. In all transfectants, class II molecules were found in endocytic compartments that labeled for CD63 and resembled the multilaminar MIIC compartments found in B cell lines. Ii is not required for endosomal targeting of class II molecules. The number of class II molecules observed in the multilaminar compartments correlates with the efficiency of antigen presentation.     

Early degenerate selection of thymocytes by class I major histocompatibility complex

Ontogeny of T cells is accomplished in the thymus by a process of positive selection, in which interaction of the T cell receptor (TcR) expressed on CD4⁺8⁺ thymocytes with self major histocompatibility complex (MHC), expressed on cortical epithelial cells, determines the progress along the maturation pathway and confers self restriction to T cells. Conversely, cells behaving as self reactive by interaction with bone marrow-derived antigen-presenting cells are negatively selected by apoptosis. We show here that the presence of a class I-restricted soluble TcR (sTcR) in the fetal thymic microenvironment, early in T cell ontogeny, determines an enhanced negative selection of a sizeable number of CD4⁺8⁺ thymocytes, which have been previously subjected to a positive-selection event. We hypothesize that the generation of the mature thymic T cell repertoire stems from an interaction of TcR, under a critical affinity threshold, with a self peptide-MHC complex which is common to a great number of TcR specificities using the same restriction element. A shift in this affinity threshold, caused by sTcR, results in the generation of cells acting in a self-reactive manner, which are then deleted. In extended fetal thymus organ culture in the presence of sTcR, we have also observed the appearance of mature CD8⁺ T cells, which once adoptively transferred to syngeneic nude mice are expanded in the periphery, consistent with an enhanced avidity of these cells for self MHC.     

Hepatitis B virus small surface antigen particles are processed in a novel endosomal pathway for major histocompatibility complex class I-restricted epitope presentation

We investigated the major histocompatibility complex (MHC) class I-restricted presentation of an epitope of the hepatitis B virus small surface (S) antigen particle to cloned murine cytotoxic T lymphocytes (CTL). Efficient L^d-restricted presentation of the S_28–39 epitope to CTL is observed in cells of different tissue origin pulsed in vitro, either with the antigenic S_28–39 12-mer S-peptide, or with particulate S-antigen. The kinetics of epitope presentation differ in S-peptide-pulsed and in S-particle-pulsed cells: while a 15-min pulse with the antigenic peptide sensitizes targets for class I-restricted CTL lysis, presentation of S-particles requires 30–60 min to sensitize cells for CTL lysis. Uptake of antigenic material and active metabolism of the presenting cell are required for processing of S-particles, but not for sensitizing targets with S-peptides. Intracellular processing and presentation of S-particles is blocked in cells treated with chloroquine, NH₄Cl, primaquine, or leupeptin, but not by treatment with cycloheximide or brefeldin A. This processing pathway operates efficiently in peptide-transporter-deficient, L^d-transfected T2 cells, revealing a novel endosomal/lysosomal processing pathway for class I-restricted presentation of peptides derived from exogenous S-particles.     

The effect of anchor residue modifications on the stability of major histocompatibility complex class I-peptide interactions

Anchor residues in peptides determine the specificity of binding to major histocompatibility complex class I molecules through interactions of their side chains with pockets in the peptide-binding groove. We have compared the kinetics of association of a Sendai virus nucleoprotein-derived peptide (FAPGNYPAL, termed SV9) with H-2K^b class I molecules, and the same peptide iodinated on the anchor residue tyrosine (¹²⁵I-SV9). Even though the association rates were too rapid for direct measurements, competition studies indicated that they were similar for SV9 and ¹²⁵I-SV9. To measure the binding of non-radioactive SV9 directly, SV9 was tritiated (³H-SV9). ³H-SV9 remained stably associated with H-2K^b molecules, whereas ¹²⁵I-SV9 dissociated in a temperature-dependent fashion. Thus, modifications on anchor residues do not necessarilly have to affect the specificity and association kinetics of peptide binding to class I molecules but can affect the stability of the resulting class I-peptide interaction. The dissociation of peptides with modified and, more generally, suboptimal anchor residue side chains may explain the presence of empty class I molecules and free class I heavy chains at the cell surface.     

Carrier-mediated uptake and presentation of a major histocompatibility complex class I-restricted peptide

Antigenic peptides derived from endogenous or viral proteins can associate with class I or class II major histocompatibility complex (MHC) molecules, while exogenous antigens are endocytosed, processed intracellularly and presented on MHC class II molecules. Here we describe a method that allows the presentation of an MHC class I-restricted antigenic peptide on MHC class I molecules, although it was taken up from the outside. The HLA-A2-restricted influenza virus matrix protein-derived peptide (flu, 57–68) was used either in soluble form or coupled via an S-S bridge to transferrin (Tf-flu). Target cells were incubated with flu or Tf-flu and the effective antigen presentation was detected in a cytotoxicity assay using flu peptide-specific, HLA-A2-restricted CD8⁺ cytotoxic T lymphocytes. Sensitization of target cells with Tf-flu required 5 to 10 times higher molar concentrations of peptide compared to sensitization with soluble free peptide. The Tf-flu construct was taken up by the cells via the Tf receptor (CD71) as the binding of Tf-flu was blocked by an excess of Tf. In contrast to the flu peptide, cytotoxicity elicited by Tf-flu was blocked by brefeldin A but not by chloroquine nor inhibitors of intracellular reducing steps, like 1-buthionine-(s, r)-sulfoximine or n-ethylmaleimide. Presentation of the flu peptide derived from Tf-flu construct is not hindered in the mutant T2 cell line, which lacks genes coding for transporter proteins for antigenic peptides (TAP1/TAP2) and proteasomes subunits, suggesting that the processing pathway described in this report may involve TAP-independent steps.     

Real-time measurement of antigenic peptide binding to empty and preloaded single-chain major histocompatibility complex class I molecules

Cytotoxic T lymphocytes (CTL) recognize peptides in association with major histocompatibility complex (MHC) class I proteins, but how peptides bind to class I is not well understood. We used a fluorescence technique to measure antigenic peptide binding to a soluble, single-chain K^d (SC-K^d) molecule in which the K^d heavy chain was connected by a 15-residue link to β₂-microglobulin. Peptides were covalently labeled at their N terminus with dansyl, and binding of dansylated K^d-restricted peptides to SC-K^d resulted in significant fluorescence enhancement, which could be inhibited by unmodified K^d-restricted peptides. Real-time binding of a dansylated peptide could be followed by monitoring the fluorescence at 530 nm. The dansylated Plasmodium berghei circumsporozoite (PbCS) 263–260 peptide bound to “empty” SC-K^d with an association rate constant of 1140 M^?1s^?1, and the subsequent spontaneous dissociation of the SC-K^d-peptide complex was slow. The dissociation increased dramatically after addition of excess unlabeled PbCS 253–260 peptide, but with a slower association constant for unlabeled peptide, 77 M^?1s^?1. Thus, the K^d-peptide complex on the surface of antigen-presenting cells should be stable, but high concentrations of peptides in the endoplasmic reticulum (ER) lumen would allow for peptide exchange on K^d before export to the surface. The apparent activation energy for PbCS 253–260 peptide binding to SC-K^d was 6.78 ± 0.64 kcal/mole, similar to values previously reported for antigen-antibody interactions.     

Major histocompatibility complex class I presentation of ovalbumin peptide 257–264 from exogenous sources: protein context influences the degree of TAP-independent presentation

Peritoneal macrophages from C57BL/6 mice process antigens from bacteria or coated on polystyrene beads for presentation by major histocompatibility complex (MHC) class I molecules. To investigate this antigen processing pathway, peritoneal macrophages from homozygous TAP1^−/− mice, which lack the transporter associated with antigen processing (TAP) and are defective in presenting endogenous antigens on MHC class I, were used. TAP1^−/− or C57BL/6 macrophages were co-incubated with either bacteria or polystyrene beads containing the 257–264 epitope from ovalbumin [OVA(257–264)], which binds the mouse class I molecule K^b. The source of the OVA(257–264) epitope was either the Crl-OVA(257–264) (Crl-OVA) fusion protein, the maltose binding protein (MBP)-Crl-OVA fusion protein, native OVA or bacterial recombinant OVA (rOVA); Crl-OVA, MBP-Crl-OVA and rOVA were each expressed in bacteria, and Crl-OVA and MBP-Crl-OVA purified from bacterial lysates and native egg OVA were coated onto polystyrene beads. The data reveal that peritoneal macrophages from C57BL/6 and TAP1^−/− mice can process bacteria expressing Crl-OVA, MBP-Crl-OVA and rOVA as well as beads coated with native OVA, purified Crl-OVA, and purified MBP-Crl-OVA and present OVA(257–264) for recognition by OVA(257–264)/K^b-specific T hybridoma cells, albeit with different relative processing efficiencies. The processing efficiency of TAP1^−/− macrophages co-incubated with bacteria or beads containing Crl-OVA or MBP-Crl-OVA was reduced approximately three to five times compared to C57BL/6 macrophages, but OVA(257–264) was presented 100 times less efficiently when the source of OVA(257–264) was full-length OVA. Chloroquine inhibition studies showed a differential requirement for acidic compartments in C57BL/6 versus TAP1^−/− macrophages, which also depended upon the source of the OVA (257–264) epitope (Crl-OVA versus full-length OVA). These data suggest that TAP1^−/− and C57BL/6 macrophages may process Crl-OVA and full-length OVA in different cellular compartments and that the protein context of the OVA(257–264) epitope influences the extent of TAP-independent processing for MHC class I presentation.     

Inhibition of natural killer cell-mediated bone marrow graft rejection by allogeneic major histocompatibility complex class I,but not class II molecules

The role of major histocompatibility complex (MHC) class I and class II molecules in natural killer (NK) cell-mediated rejection of allogeneic, semi-syngeneic and MHC-matched bone marrow grafts was investigated. The use of β₂-microglobulin (β₂m) -/- and β₂m +/- mice as bone marrow donors to MHC-mismatched recipients allowed an analysis of whether the presence of semi-syngeneic and allogeneic MHC class I gene products would be triggering, protective or neutral, in relation to NK cell-mediated rejection. Loss of β₂m did not allow H-2^b bone marrow cells to escape from NK cell-mediated rejection in allogeneic (BALB/c) or semi-allogeneic (H-2D^d transgenic C57BL/6) mice. On the contrary, it led to stronger rejection, as reflected by the inability of a larger bone marrow cell inoculum to overcome rejection by the H-2-mismatched recipients. In H-2-matched recipients, loss of β₂m in the graft led to a switch from engraftment to rejection. At the recipient level, loss of β₂m led to loss of the capability to reject H-2-matched β₂m-deficient as well as allogeneic grafts. When MHC class II-deficient mice were used as donors, the response was the same as that against donors of normal MHC phenotype: allogeneic and semi-syngeneic grafts were rejected by NK cells, while syngeneic grafts were accepted. These data suggest a model in which allogeneic class I molecules on the target cell offer partial protection, while certain syngeneic class I molecules give full protection from NK cell-mediated rejection of bone marrow cells. There was no evidence for a role of MHC class II molecules in this system.     

Assembly of an abundant endogenous major histocompatibility complex class II/peptide complex in class II compartments

To identify the intracellular site(s) of formation of an endogenous class II/peptide complex in a human B cell line, we employed kinetic pulse-chase labeling experiments followed by subcellular fractionation by Percoll density gradient centrifugation and immunogold labeling on ultrathin cryosections. For direct demonstration of assembly of such complexes, we used the monoclonal antibody YAe, which detects an endogenous complex of the mouse class II molecule I-A^b with a 17-amino acid peptide derived from the α chain of HLA-DR (DRα52–68). We show that in human B lymphocytes, these class II/peptide complexes assemble and transiently accumulate in major histocompatibility complex class II-enriched compartments before reaching the cell surface.     

Trypanosoma cruzi-infected macrophages are defective in major histocompatibility complex class II antigen presentation

Trypanosoma cruzi, the intracellular protozoan parasite that causes Chagas' disease, interferes with the host immune response to establish a persistent infection. In this report, we demonstrate that macrophages infected with T. cruzi are unable to effectively present antigens to CD4 T cells. The interference is due to defective antigen-presenting cell (APC) function, as antigen-independent stimulation of the T cell in the presence of infected macrophages is not affected. The defect is distal to antigen processing and is not due to decreased major histocompatibility complex (MHC) class II expression, decreased viability, defective peptide loading in the infected macrophages, nor absence of CD28 co-stimulation. There was a role for gp39:CD40 co-stimulation during antigen presentation to the T cells we studied, but the expression of CD40 on T. cruzi-infected macrophages was not decreased. Antigen-specific adhesion between macrophages and T cells was reduced by infection. Equivalent levels of the adhesion molecules lymphocyte function-associated antigen-1, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 or very late antigen-4 are found on infected and uninfected APC, suggesting that reduced expression of these adhesion molecules was not responsible for the defect in antigen-specific adhesion. The defective T cell:macrophage adhesion may be due to the reduced expression of other adhesion molecules or other changes in the cell induced by infection. Interfering with MHC class II antigen presentation in infected macrophages may help T. cruzi to blunt the immune response by the host.     

Genes encoded in the major histocompatibility complex affecting the generation of peptides for TAP transport

The B cell line 721.174 has lost the ability to present intracellular antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). This phenotype results from a homozygous deletion in the MHC that includes the peptide transporter genes TAP1 and TAP2, and the proteasome subunits LMP2 and LMP7. Recent work has shown that such cells transfected with TAP genes load their class I molecules with endogenous peptides, and present several viral epitopes to class I-restricted CTL. These data implied that the LMP2 and LMP7 genes were not required for the presentation of most epitopes through class I molecules. By contrast, while confirming the previous reports, we have identified several epitopes that appear to require genes in the MHC in addition to the TAP for their presentation. Further analysis localizes the defect to proteolysis in the cytosol. In one case, presentation could be partially restored by re-expression of full-length LMP7. Control experiments with LMP7, from which the putative pro-region had been removed, failed to restore presentation, and this lack of effect correlated with failure of the shortened LMP7 to incorporate into the proteasome. These results suggest a role for LMP7 in the generation of a viral epitope, but leave open the possibility that additional genes within the .174 deletion are required for full restoration of antigen presentation.