Inhibition of class I and class II MHC-restricted antigen presentation by cytotoxic T lymphocytes specific for an exogenous antigen.

Ag in the extracellular fluids can be internalized, processed, and presented in association with class I MHC molecules on specialized APC in normal spleen. We examine the fate of these APC after they present Ag to a CTL. When splenocytes present exogenous OVA to CTL, their ability to subsequently present native Ag in association with both class I and class II molecules is inhibited. CTL do not inhibit the ability of splenocytes to present processing independent peptides with class I or class II molecules. Inhibition of Ag presentation is only observed in the presence of the specific Ag recognized by the CTL. This inhibition is MHC-restricted. In the presence of specific Ag, CTL inhibit the ability of APC to present unrelated Ag. However, bystander APC are not affected by activated CTL. Taken together these results indicate that when APC present exogenous Ag to CTL, they are inhibited or killed. The CTL that mediates this activity has a conventional CD4-CD8+ phenotype and utilizes a TCR-alpha beta. The potential significance of these findings and their possible relationship to phenomena associated with Ts cells are discussed.     

ATGs help MHC class II,but inhibit MHC class I antigen presentation

We have recently shown that the LC3/Atg8 lipidation machinery of macroautophagy is involved in the internalization of MHC class I molecules. Decreased internalization in the absence of ATG5 or ATG7 leads to MHC class I surface stabilization on dendritic cells and macrophages, resulting in elevated CD8⁺ T cell responses during viral infections and improved immune control. Here, we discuss how the autophagic machinery supports MHC class II restricted antigen presentation, while compromising MHC class I presentation via internalization and degradation.     

Calcineurin inhibitors block MHC-restricted antigen presentation in vivo

APCs, like T cells, are affected by calcineurin inhibitors. In this study, we show that calcineurin inhibitors efficiently block MHC-restricted exogenous Ag presentation in vivo. Mice were injected with clinical doses of tacrolimus (FK-506) followed by soluble OVA, and dendritic cells (DCs) were isolated from lymph nodes and spleens. The efficacy of OVA peptide presentation by DCs was evaluated using OVA-specific CD8 and CD4 T cells. Tacrolimus inhibited both class I- and class II-restricted DC presentation of OVA to T cells. Tacrolimus also inhibited both class I- and class II-restricted presentation of OVA in peritoneal macrophages isolated from mice injected with tacrolimus followed by soluble OVA. Tacrolimus-treated peritoneal macrophages, however, were able to present synthetic OVA peptide, SIINFEKL. Inclusion of cyclosporine A to biodegradable microspheres containing OVA greatly reduced their capacity to induce OVA-specific CTL response in mice. These findings provide novel insight into the mode of action of calcineurin inhibitors and have important implications for clinical immunosuppression regimens.     

Proteasome and class I antigen processing and presentation

The recent discovery of two proteasome homologous genes,LMP2 andLMP7, in the class II region of the human MHC, has implicated this multi-subunit protease in an early step of the immune response; the degradation of intracellular and viral proteins. Short peptides produced by the proteasome are transported into the ER by the product of another set of MHC class II genes,TAP1 andTAP2, where they bind and stabilise HLA class I molecules. Antigenic peptides displayed at the cell surface by HLA class I molecules mark cells for destruction by cytotoxic T lymphocytes. The role of the proteasome in antigen processing was questioned when mutant cells, which lack theLMP genes, were able to process and present antigens normally. The discovery that two proteasome -subunits, delta andMB1, highly homologous toLMP2 andLMP7 and expressed in reciprocal manner, is now consistent with a role for the proteasome in antigen processing. The incorporation of different -subunits into the proteasome may be a mechanism to modulate catalytic activity of the proteasome complex, allowing production of peptides that are more suitable to enter into the ER by the TAP transporters and to bind HLA class I molecules. But, in the absence of the LMPs, the other subunits permit processing of most antigens reasonably efficiently.Abbreviations ABC ATP-binding cassete - 2m 2-microglobulin - ER endoplasmic reticulum - IFN interferon - LMP low molecular weight peptide - MHC major histocompatibility complex - TAP transporter associated with antigen processing     

Generation of class I MHC-restricted T-T hybridomas

In this report we describe a system for the generation of functional, class I MHC-restricted, T-T hybridomas. The BW5147 cell line was transfected with the CD8 gene. BW5147 transfectants were obtained that stably expressed CD8 and this expression was maintained after somatic cell hybridization with activated T lymphocytes. To determine whether the stable expression of CD8 would facilitate the generation of class I MHC-specific T-T hybridomas, the transfected cells were fused with alloreactive T cells and the resultant hybrids were screened for their ability to produce lymphokines in response to antigenic stimulation. Somatic cell hybridizations with BW5147-CD8 transfectants give rise to a much higher frequency of class I MHC-specific T-T hybridomas relative to parallel fusions with BW5147. To determine whether the BW5147-CD8 transfectants would also support the generation of Ag-specific, class I MHC-restricted T-T hybridomas, they were fused with OVA-specific CTL. Several T-T hybrid clones were identified that produced lymphokines after stimulation with a transfected APC that was synthesizing OVA, or with a tryptic digest of OVA in the presence of syngeneic APC. The stimulation by Ag was MHC-restricted and mapped to the Kb molecule. An anti-CD8 mAb inhibited the stimulation of these hybridomas by Ag plus APC, whereas their stimulation by mitogen was unaffected. Cytolytic activity was not detected when several of the OVA-specific or alloreactive hybridomas were tested for their ability to kill target cells bearing the appropriate Ag. These results demonstrate that the BW5147-CD8 transfectants allow the generation of class I MHC-restricted T-T hybridomas. The potential utility of this system is discussed.     

Roles for asparagine endopeptidase in class II MHC-restricted antigen processing

The adaptive immune response depends on the creation of suitable peptides from foreign antigens for display on MHC molecules to T lymphocytes. Similarly, MHC-restricted display of peptides derived from self proteins results in the elimination of many potentially autoreactive T cells. Different proteolytic systems are used to generate the peptides that are displayed as T cell epitopes on class I compared with class II MHC molecules. In the case of class II MHC molecules, the proteases that reside within the endosome/lysosome system of antigen-presenting cells are responsible; surprisingly, however, there are relatively few data on which enzymes are involved. Recently we have asked whether proteolysis is required simply in a generic sense, or whether the action of particular enzymes is needed to generate specific class II MHC-associated T cell epitopes. Using the recently identified mammalian asparagine endopeptidase as an example, we review recent evidence that individual enzymes can make clear and non-redundant contributions to MHC-restricted peptide display.     

Tat-mediated protein delivery can facilitate MHC class I presentation of antigens

We have previously shown that the tat protein of HIV-1 can be used as a carrier to promote the intracellular delivery of heterologous proteins. Here we have tested if the tat-delivery technology can be used to direct MHC class I presentation of native protein, using ovalbumin (OVA) as a model system. We show that a tat-ovalbumin conjugate (tatOVA) can be delivered into cells and that subsequent processing and presentation occurs, resulting in effective and specific killing of these target cells by an OVA specific cytotoxic T-lymphocyte (CTL) line. Comparison with the E.G7 line that expresses the OVA gene indicates that tat-mediated delivery is as efficient as endogenous expression in this system. Tat-mediated antigenic protein delivery may be useful both as a research technique and, potentially, as a therapeutic or prophylactic vaccine.     

Macrophage-colony stimulating factor enhances MHC-restricted presentation of exogenous antigen in dendritic cells

Previous studies have shown that dendritic cells (DCs) can phagocytize, process and present a microencapsulated form of ovalbumin (OVA) in the context of class I MHC as well as class II MHC. In the present study, we examined the effects of recombinant human macrophage-colony stimulating factor (M-CSF) on the MHC-restricted presentation of microencapsulated OVA by DCs. Two types of DCs were generated from mouse bone marrow (BM) cells, one type with granulocyte/macrophage-colony stimulating factor (GM-CSF) alone, the other type with GM-CSF and interleukin (IL)-4. Pretreatment with M-CSF significantly enhanced both class I MHC and class II MHC-restricted presentation of exogenous OVA by both types of DCs. The enhancing activity of M-CSF on antigen presentation was more potent in DCs generated with GM-CSF alone compared to DCs generated with both GM-CSF and IL-4. Pretreatment of the DCs with M-CSF did not increase phagocytic activity or total level of expression of class I MHC (H-2K(b)) molecules, but increased expression of OVA peptide-H-2K(b) complexes upon phagocytosis of microencapsulated OVA. These results demonstrate that M-CSF increases intracellular processing events of phagocytized antigen in DCs.     

Towards a systems understanding of MHC class I and MHC class II antigen presentation

The molecular details of antigen processing and presentation by MHC class I and class II molecules have been studied extensively for almost three decades. Although the basic principles of these processes were laid out approximately 10 years ago, the recent years have revealed many details and provided new insights into their control and specificity. MHC molecules use various biochemical reactions to achieve successful presentation of antigenic fragments to the immune system. Here we present a timely evaluation of the biology of antigen presentation and a survey of issues that are considered unresolved. The continuing flow of new details into our understanding of the biology of MHC class I and class II antigen presentation builds a system involving several cell biological processes, which is discussed in this Review.     

Toxoplasma gondii and MHC-restricted antigen presentation: on degradation, transport and modulation

Resistance against Toxoplasma gondii, an obligate intracellular protozoan parasite surrounded by a parasitophorous vacuolar membrane, is mediated by the cellular arm of the immune system, namely CD8+ and CD4+ T cells. Thus, priming and activation of these cells by presentation of antigenic peptides in the context of major histocompatibility complex class I and class II molecules have to take place. This is despite the fact that the vacuolar membrane avoids fusion with the endocytic compartment and acts like a molecular sieve, restricting passive diffusion of larger molecules. This raises several cell biological and immunological questions which will be discussed in this review in the context of our current knowledge about major histocompatibility complex-restricted antigen presentation in other systems: (1) By which pathways are parasite-derived antigens presented to T cells? (2) Has the parasite evolved mechanisms to interfere with major histocompatibility complex-restricted antigen presentation in order to avoid immune recognition? (3) To what extent and by which mechanism is antigenic material, originating from the parasite, able to pass through the vacuolar membrane into the cytosol of the infected cell and is it then accessible to the antigen presentation machinery of the infected cell? (4) What are the actual antigen-presenting cells which prime specific T cells in lymphoid organs? An understanding of these mechanisms will not only provide new insights into the pathogenesis of Toxoplasma gondii and possibly other intravacuolar parasites, but will also improve vaccination strategies.     

Peptidomimetic compounds that inhibit antigen presentation by autoimmune disease-associated class II major histocompatibility molecules.

We have identified a heptapeptide with high affinity to rheumatoid arthritis-associated class II major histocompatibility (MHC) molecules. Using a model of its interaction with the class II binding site, a variety of mimetic substitutions were introduced into the peptide. Several unnatural amino acids and dipeptide mimetics were found to be appropriate substituents and could be combined into compounds with binding affinities comparable to that of the original peptide. Compounds were designed that were several hundred-fold to more than a thousand-fold more potent than the original peptide in inhibiting T-cell responses to processed protein antigens presented by the target MHC molecules. Peptidomimetic compounds of this type could find therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of autoimmune diseases.     

Intracellular Salmonella inhibit antigen presentation by dendritic cells

Dendritic cells (DC) are important APCs linking innate and adaptive immunity. During analysis of the intracellular activities of Salmonella enterica in DC, we observed that viable bacteria suppress Ag-dependent T cell proliferation. This effect was dependent on the induction of inducible NO synthase by DC and on the function of virulence genes in Salmonella pathogenicity island 2 (SPI2). Intracellular activities of Salmonella did not affect the viability, Ag uptake, or maturation of DC, but resulted in reduced presentation of antigenic peptides by MHC class II molecules. Increased resistance to reinfection was observed after vaccination of mice with SPI2-deficient Salmonella compared with mice vaccinated with SPI2-proficient Salmonella, and this correlated with an increased amount of CD4(+) as well as CD8(+) T cells. Our study is the first example of interference of an intracellular bacterial pathogen with Ag presentation by DC. The subversion of DC functions is a novel strategy deployed by this pathogen to escape immune defense, colonize host organs, and persist in the infected host.     

1st class ticket to class I: protein toxins as pathfinders for antigen presentation

A number of bacterial toxins have evolved diverse strategies for crossing membrane barriers in order to reach their substrates in the mammalian cytosol. Recent studies show that this property can be exploited for the delivery of fused antigens into the major histocompatibility complex class I-restricted presentation pathway, with the goal of eliciting a specific immune response. Here we discuss the peculiarities of the trafficking pathways of a variety of toxins, and how these may allow the toxins to be used as delivery vehicles for therapeutic and diagnostic purposes.     

Recognition of the influenza hemagglutinin by class II MHC-restricted T lymphocytes and antibodies. I. Site definition and implications for antigen presentation and T lymphocyte recognition.

We have identified the site encompassing residues 126-145 on the A/Japan/57 influenza hemagglutinin molecule that is recognized in association with HLA-DRw11 by a clonal population of human, influenza specific, CD4+ cytolytic T lymphocytes. The critical core sequence of the T cell determinant spans hemagglutinin residues 129-140 and overlaps a putative antibody binding site. Hemagglutinins of influenza field strains that are not recognized by the T cell clones contain sequence alterations within the 129-140 target site of the CD4+ T cells. Functional analyses, with synthetic peptides, of the contribution of each of the residues within the sequence toward the capacity of the antigenic fragment to associate with both the restriction element and the TCR revealed a continuous linear array of residues necessary for MHC binding and/or Ag receptor engagement. At least one residue, the lysine at position 134, was shown to be critical for both DRw11 association and TCR recognition. The significance of these findings for recognition of glycoproteins by human CD4+ T cells is discussed.     

Immunosuppressive effects of polyunsaturated fatty acids on antigen presentation by human leukocyte antigen class I molecules

Dietary supplementation with polyunsaturated fatty acids (PUFAs) has immunosuppressive effects; however, the molecular targets of PUFAs and their mode of action remain unclear. One possible target is antigen presentation to T cells through the human leukocyte antigen (HLA) class I pathway. Here we show that incorporation of PUFAs lowers target cell susceptibility to lysis by effector T cells. Treatment of B lymphoblast targets with the omega-6 PUFA arachidonic acid (AA) or omega-3 docosahexaenoic acid lowered their susceptibility to lysis by alloreactive CD8+ T cells by approximately 20-25%. HLA class I surface levels and their rate of endoplasmic reticulum (ER)-Golgi traffic were also reduced by PUFA treatment. Calibration experiments showed that the approximately 15% reduction in surface HLA I was not sufficient to completely account for the decreased lysis. However, PUFAs significantly lowered antigen-presenting cell-T cell conjugate formation, by approximately 30-40%. Taken together, our data show for the first time that an omega-6 and an omega-3 PUFA affect the HLA class I pathway of B lymphoblasts. Our findings suggest that elimination of self- and pathogen-derived peptides by effectors may be compromised by dietary PUFA supplementation. In addition, PUFA-mediated changes in ER-Golgi trafficking point to a new area of PUFA modulation of immune responses.     

Macrophages as accessory cells for class I MHC-restricted immune responses.

Ag do not elicit T lymphocyte responses unless they are presented in conjunction with MHC molecules on the surface of an appropriate APC. In the case of CD4+ T lymphocytes dendritic cells can deliver all signals required for complete induction as can macrophages and (activated) B cells. The function of CTL also depends on the presence of specialized accessory cells. Here we show that these accessory cells can behave like scavenger cells. They use foreign Ag in the form of cellular debris as immunogen. They are also crucial for CTL induction because in vivo depletion of phagocytotic cells completely inhibits CTL responses. In these animals CTL activity could be restored by transfer of macrophages. All of the reappearing CTL used MHC restriction elements expressed by the infused macrophages. These experiments suggest that a cognate interaction between macrophages and CTL precursors initiates class I MHC-restricted immune responses.     

Cutting Edge: Selective role of ubiquitin in MHC class I antigen presentation

The importance of ubiquitination in MHC class I-restricted Ag processing remains unclear. To address this issue, we overexpressed wild-type and dominant-negative lysineless forms of ubiquitin (Ub) in mammalian cells using an inducible vaccinia virus system. Overexpression of the lysineless Ub nearly abrogated polyubiquitination and potently inhibited epitope presentation from a cytosolic N-end rule substrate as well as endoplasmic reticulum (ER)-targeted model Ags. In contrast, there was little impact on Ag presentation from cytosolic proteins. These trends were location dependent; redirecting cytosolic Ag to the ER rendered presentation lysineless Ub-sensitive, whereas retargeting exocytic Ag to the cytosol had the inverse effect. This dichotomy was further underscored by small interfering RNA knockdown of the ER-associated Ub ligase Hrd1. Thus, Ub-dependent degradation appears to play a major role in the MHC class I-restricted processing of ER-targeted proteins and a more restricted role in the processing of cytosolic proteins.     

Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals

Genetic modification of vaccines by linking the Ag to lysosomal or endosomal targeting signals has been used to route Ags into MHC class II processing compartments for improvement of CD4+ T cell responses. We report in this study that combining an N-terminal leader peptide with an MHC class I trafficking signal (MITD) attached to the C terminus of the Ag strongly improves the presentation of MHC class I and class II epitopes in human and murine dendritic cells (DCs). Such chimeric fusion proteins display a maturation state-dependent subcellular distribution pattern in immature and mature DCs, mimicking the dynamic trafficking properties of MHC molecules. T cell response analysis in vitro and in mice immunized with DCs transfected with Ag-encoding RNA showed that MITD fusion proteins have a profoundly higher stimulatory capacity than wild-type controls. This results in efficient expansion of Ag-specific CD8+ and CD4+ T cells and improved effector functions. We used CMVpp65 and NY-ESO-1 Ags to study preformed immune responses in CMV-seropositive individuals and cancer patients. We show that linking these Ags to the MITD trafficking signal allows simultaneous, polyepitopic expansion of CD8+ and CD4+ T cells, resulting in distinct CD8+ T cell specificities and a surprisingly broad and variable Ag-specific CD4+ repertoire in different individuals.