Presentation of Phagocytosed Antigens by MHC Class I and II

Phagocytosis provides innate immune cells with a mechanism to take up and destroy pathogenic bacteria, apoptotic cells and other large particles. In some cases, however, peptide antigens from these particles are preserved for presentation in association with major histocompatibility complex (MHC) class I or class II molecules in order to stimulate antigen‐specific T cells. Processing and presentation of antigens from phagosomes presents a number of distinct challenges relative to antigens internalized by other means; while bacterial antigens were among the first discovered to be presented to T cells, analyses of the cellular mechanisms by which peptides from phagocytosed antigens assemble with MHC molecules and by which these complexes are then expressed at the plasma membrane have lagged behind those of conventional model soluble antigens. In this review, we cover recent advances in our understanding of these processes, including the unique cross‐presentation of phagocytosed antigens by MHC class I molecules, and in their control by signaling modalities in phagocytic cells.     

Structural Mechanism of ER Retrieval of MHC Class I by Cowpox

One of the hallmarks of viral immune evasion is the capacity to disrupt major histocompatibility complex class I (MHCI) antigen presentation to evade T-cell detection. Cowpox virus encoded protein CPXV203 blocks MHCI surface expression by exploiting the KDEL-receptor recycling pathway, and here we show that CPXV203 directly binds a wide array of fully assembled MHCI proteins, both classical and non-classical. Further, the stability of CPXV203/MHCI complexes is highly pH dependent, with dramatically increased affinities at the lower pH of the Golgi relative to the endoplasmic reticulum (ER). Crystallographic studies reveal that CPXV203 adopts a beta-sandwich fold similar to poxvirus chemokine binding proteins, and binds the same highly conserved MHCI determinants located under the peptide-binding platform that tapasin, CD8, and natural killer (NK)-receptors engage. Mutagenesis of the CPXV203/MHCI interface identified the importance of two CPXV203 His residues that confer low pH stabilization of the complex and are critical to ER retrieval of MHCI. These studies clarify mechanistically how CPXV203 coordinates with other cowpox proteins to thwart antigen presentation.     

P2X7 Receptor Activation Impairs Exogenous MHC Class I Oligopeptides Presentation in Antigen Presenting Cells

Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8⁺ T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5′-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8⁺ T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8⁺ T cell immunity.     

Intracellular Assembly and Trafficking of MHC Class I Molecules

The presentation of antigenic peptides by class I molecules of the major histocompatibility complex begins in the endoplasmic reticulum (ER) where the co-ordinated action of molecular chaperones, folding enzymes and class I-specific factors ensures that class I molecules are loaded with high-affinity peptide ligands that will survive prolonged display at the cell surface. Once assembled, class I molecules are released from the quality-control machinery of the ER for export to the plasma membrane where they undergo dynamic endocytic cycling and turnover. We review recent progress in our understanding of class I assembly, anterograde transport and endocytosis and highlight some of the events targeted by viruses as a means to evade detection by cytotoxic T cells and natural killer cells.     

Coevolution between MHC Class I and Antigen-Processing Genes in Salamanders

Proteins encoded by antigen-processing genes (APGs) provide major histocompatibility complex (MHC) class I (MHC-I) with antigenic peptides. In mammals, polymorphic multigenic MHC-I family is served by monomorphic APGs, whereas in certain nonmammalian species both MHC-I and APGs are polymorphic and coevolve within stable haplotypes. Coevolution was suggested as an ancestral gnathostome feature, presumably enabling only a single highly expressed classical MHC-I gene. In this view coevolution, while optimizing some aspects of adaptive immunity, would also limit its flexibility by preventing the expansion of classical MHC-I into a multigene family. However, some nonmammalian taxa, such as salamanders, have multiple highly expressed MHC-I genes, suggesting either that coevolution is relaxed or that it does not prevent the establishment of multigene MHC-I. To distinguish between these two alternatives, we use salamanders (30 species from 16 genera representing six families) to test, within a comparative framework, a major prediction of the coevolution hypothesis: the positive correlation between MHC-I and APG diversity. We found that MHC-I diversity explained both within-individual and species-wide diversity of two APGs, TAP1 and TAP2, supporting their coevolution with MHC-I, whereas no consistent effect was detected for the other three APGs (PSMB8, PSMB9, and TAPBP). Our results imply that although coevolution occurs in salamanders, it does not preclude the expansion of the MHC-I gene family. Contrary to the previous suggestions, nonmammalian vertebrates thus may be able to accommodate diverse selection pressures with flexibility granted by rapid expansion or contraction of the MHC-I family, while retaining the benefits of coevolution between MHC-I and TAPs.     

Activation of human T4 cells by cross-linking class I MHC molecules

These studies examined whether cross-linking class I MHC molecules results in functional or biochemical responses in human T4 cells. The initial studies demonstrated that cross-linking class I MHC molecules either by culturing highly purified T4 cells with immobilized mAb to class I MHC Ag or reacting the T4 cells with mAb to class I MHC Ag and then cross-linking the mAb with goat antimouse Ig (GaMIg) enhanced T4 cell proliferation induced by an immobilized mAb to CD3, OKT3. More-over, immobilized but not soluble mAb to class I MHC Ag enhanced T4 cell proliferation induced by the combination of two mAb to CD2, OKT11, and D66.2. Finally, T4 cells reacted with mAb to CD3 and class I MHC Ag proliferated in the presence of IL-2 when cross-linked with GaMIg more vigorously than T4 cells reacted with either mAb alone. Cross-linking class I MHC molecules was also found to stimulate T4 cells directly. T4 cells reacted with mAb to class I MHC Ag or beta 2 microglobulin and cross-linked with GaMIg proliferated vigorously in the presence of IL-2 or PMA. In addition, it was demonstrated that cross-linking class I MHC molecules by culturing T4 cells with immobilized mAb to class I MHC Ag induced T4 cell proliferation in the presence of IL-2. T4 cell proliferation in the presence of IL-2 and PMA could also be induced by reacting the cells with specific mAb to polymorphic determinants on class I MHC molecules and cross-linking with GaMIg. Cross-linking mAb to CD4 or CD11a did not have a similar functional effect on T4 cells. Finally it was demonstrated that adding GaMIg to T4 cells reacted with mAb to class I MHC Ag but not CD11a resulted in an increase in intracellular calcium concentration. The data demonstrate that cross-linking class I MHC molecules results in the generation of at least one activation signal, a rise in intracellular calcium concentration, and, thereby, stimulates human T4 cells.     

Characterization and Phylogenetic Relationship of Prosimian MHC Class I Genes

MHC class I cDNA sequences from the most divergent primate group of extant primates compared to human, the suborder Strepsirrhini (prosimians), are described. The sequences are derived from the gray mouse lemur (Microcebus murinus) and the ring-tailed lemur (Lemur catta), which are members of the malagasy Lemuriformes, as well as from the pygmy slow loris (Nycticebus pygmaeus), a prosimian from East Asia. The M. murinus sequences have been analyzed in detail. Analysis of the expression level, G/C content, and synonymous vs. nonsynonymous substitution rates in the peptide-binding region codons suggests that these cDNA clones represent classical class I (class Ia) genes. According to Southern blot analysis, the genome of the gray mouse lemur might contain about 10 class I genes. In gene tree analysis, the strepsirrhine class Ia genes described here cluster significantly separately from the known class I genes of Catarrhini (humans, apes, Old World monkeys) and Platyrrhini (New World monkeys) species, suggesting that the class I loci of Simiiformes arose by gene duplications which occurred after the divergence of prosimians.     

Comparison of rat MHC class I antigens by peptide mapping

Monoclonal antibodies specific for the rat major histocompatibility complex (MHC) class I antigens RT1.Aⁿ, RT1.A^u, and RT1.E^u were used for immunoprecipitation of antigens biosynthetically radiolabeled with¹⁴C- or³H-labeled arginine, lysine, and tyrosine; with arginine or tyrosine alone; and with or without tunicamycin in the culture medium. Heavy chains of the glycosylated and unglycosylated antigens were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their tryptic and chymotryptic peptides were compared by high performance liquid chromatography. The antigens coded by the same locus in two different haplotypes (Aⁿ and A^u) differed by 30%, whereas the products of two different loci in the same haplotype (A^u and E^u) differed only by 1–3%. Comparative analysis of the data for samples labeled with single amino acids indicated that two amino acids in A^u have been substituted by an arginine and probably by a tyrosine residue, respectively, in E^u. The high degree of homology between the products of theA andE loci in the same haplotype accounts for the difficulty in detecting recombinational events within the MHC of the rat by classical serological approaches.We dedicate this publication to Professor Paul Doty on the occasion of his sixty-fifth birthday     

Class I MHC is stabilized against thermal denaturation by physiological concentrations of NaCl

Class I MHC molecules are ternary complexes composed of an allotype specific heavy chain, a noncovalently associated protein beta(2)-microglobulin (beta(2)m), and a peptide. The complexes are assembled in the endoplasmic reticulum by a complex series of chaperones and peptide-loading mechanisms. In the absence of beta(2)m or peptide, very little class I heavy chain is transported to the surface of the cell. Complexes that do not contain all three parts of the protein are not made productively in vivo and not at all in vitro. The ability of the complex to withstand thermal denaturation in vitro has been shown to be related to the binding affinity of the peptide. Paradoxically, some low-affinity peptide complexes denature at or below human basal body temperatures in vitro but are effective biological agents in vivo. Here we show that these complexes are stabilized against thermal denaturation by physiological cosolvents and maximally stabilized by 150 mM NaCl. While the degree of stabilization by 150 mM NaCl is greatest for low-affinity peptide/MHC complexes, the mechanism of stabilization is independent of peptide sequence. This effect is hypothesized to occur by multiple mechanisms including increasing the affinity of beta(2)m for the complex and charge screening.     

Analysis of class I MHC antigens in the rat by monoclonal antibodies

Monoclonal antibodies (mAb) were made against class I MHC antigens of the i (mAb 42,70,39) and u (mAb 68-D) haplotypes in the rat by using specific strain combinations in order to obtain reagents for identifying the products of the RT1.An, RT1.Au, and RT1.Eu loci. These antibodies were hemagglutinating only; were IgG except for mAb 68-D3, which had a defective heavy chain; reacted identically with MHC-congenic strains and with their inbred donor strains; and precipitated class I MHC antigens. Strain distribution, sequential immunoprecipitation, and peptide mapping studies were used to define the specificities of the mAb, and the assignments were checked by comparing the specificities of the mAb with those of haplotype-specific alloantisera. The specificities were the following: mAb 42, An; mAb 68-D, Au; mAb 70, Eu; and mAb 39, an antigen encoded by a locus different from A and E. This new locus was designated RT1.F, and the allele detected by mAb 39, as Fa. The serologic data place RT1.F between RT1.A and RT1.D. The plasma membranes of DA.1I(BI) lymphocytes contain comparable amounts of An, Eu, and Fa antigens but express them on the cell surface in the order An much greater than Eu greater than Fa.     

Activation of human T cell clones and Jurkat cells by cross-linking class I MHC molecules

Cross-linking class I MHC molecules on human T cell clones by reacting them with various mAb directed at either monomorphic or polymorphic determinants on class I MHC molecules followed by cross-linking with GaMIg stimulated a rise in intracellular free calcium concentration ([Ca2+]i), and induced proliferation and IL-2 production. T cell clones varied in the mean density of class I MHC molecules and the capacity to respond to mAb to class I MHC molecules. However, the functional responses of the clones did not correlate with class I MHC density or the CD4/CD8 phenotype. mAb to polymorphic class I MHC determinants were less able to induce an increase in [Ca2+]i and a functional response in the T cell clones. Additive stimulatory effects were noted when mAb against both HLA-A and HLA-B determinants were employed. Cross-linking class I MHC molecules on Jurkat cells induced a rise by [Ca2+]i and induced IL-2 production upon co-stimulation with PMA. Cross-linking class I MHC molecules on mutant Jurkat cells that expressed diminished levels of CD3 and were unable to produce IL-2 in response to anti-CD3 stimulation triggered both a rise in [Ca2+]i and IL-2 production with PMA co-stimulation. In contrast, cross-linking class I MHC molecules on mutant Jurkat cells that were CD3- stimulated neither a rise in [Ca2+]i nor IL-2 production. The combination of mAb to CD28 or ionomycin and PMA, however, was able to induce IL-2 production by CD3- Jurkat cells. The data demonstrate that cross-linking class I MHC molecules delivers a functionally important signal to T cell clones and Jurkat cells and indicate that class I MHC molecules may function to transduce activation signals to T cells. In addition, the data demonstrate that transmission of an activation signal via class I MHC molecules requires CD3 expression. The data, therefore, support a central role for CD3 in the transduction of activation signals to T cells via class I MHC molecules.     

Suppression of the Macrophage Proteasome by Ethanol Impairs MHC Class I Antigen Processing and Presentation

Alcohol binge-drinking (acute ethanol consumption) is immunosuppressive and alters both the innate and adaptive arms of the immune system. Antigen presentation by macrophages (and other antigen presenting cells) represents an important function of the innate immune system that, in part, determines the outcome of the host immune response. Ethanol has been shown to suppress antigen presentation in antigen presenting cells though mechanisms of this impairment are not well understood. The constitutive and immunoproteasomes are important components of the cellular proteolytic machinery responsible for the initial steps critical to the generation of MHC Class I peptides for antigen presentation. In this study, we used an in-vitro cell culture model of acute alcohol exposure to study the effect of ethanol on the proteasome function in RAW 264.7 cells. Additionally, primary murine peritoneal macrophages obtained by peritoneal lavage from C57BL/6 mice were used to confirm our cell culture findings. We demonstrate that ethanol impairs proteasome function in peritoneal macrophages through suppression of chymotrypsin-like (Cht-L) proteasome activity as well as composition of the immunoproteasome subunit LMP7. Using primary murine peritoneal macrophages, we have further demonstrated that, ethanol-induced impairment of the proteasome function suppresses processing of antigenic proteins and peptides by the macrophage and in turn suppresses the presentation of these antigens to cells of adaptive immunity. The results of this study provide an important mechanism to explain the immunosuppressive effects of acute ethanol exposure.     

Prediction of MHC Class I Binding Peptides by a Query Learning Algorithm Based on Hidden Markov Models

A query learning algorithm based on hidden Markov models (HMMs) isdeveloped to design experiments for string analysis and prediction of MHCclass I binding peptides. Query learning is introduced to aim at reducingthe number of peptide binding data for training of HMMs. A multiple numberof HMMs, which will collectively serve as a committee, are trained withbinding data and used for prediction in real-number values. The universeof peptides is randomly sampled and subjected to judgement by the HMMs.Peptides whose prediction is least consistent among committee HMMs aretested by experiment. By iterating the feedback cycle of computationalanalysis and experiment the most wanted information is effectivelyextracted. After 7 rounds of active learning with 181 peptides in all,predictive performance of the algorithm surpassed the so far bestperforming matrix based prediction. Moreover, by combining the bothmethods binder peptides (log Kd < -6) could be predicted with84% accuracy. Parameter distribution of the HMMs that can be inspectedvisually after training further offers a glimpse of dynamic specificity ofthe MHC molecules.     

Class I MHC allochimeric presentation of composite immunogenic and self epitopes induces tolerance to genetically diverse rat strains

Functional topography of rat class I major histocompatibility complex (MHC) molecule was studied. The α₁-helical sequences that are shared by class I RT1.A^l and RT1.A^u were substituted in the RT1.A^a molecule to produce the composite -RT1.A^a MHC class I allochimeric molecule. Dominant immunogenic epitopes that induce accelerated rejection were identified within the hypervariable regions of the α₁ domain of RT1.A^a, RT1.A^l, and RT1.A^u. Peri-transplant portal venous delivery of MHC class I allochimeric proteins, that included composite α₁ helical immunodominant epitopes of RT1.A^u and RT1.A^l, induced donor-specific tolerance to RT1^u (Wistar Furth, WF) and RT1^l Lewis, LEW) disparate cardiac allografts in ACI (RT1^a) hosts. Allochimeric generated tolerance was characterized by absence of T cell deletion or anergy. Donor specific IgM allo-Abs was not detected, while IgG alloresponse was markedly attenuated in sera of tolerant hosts. Further, long-term allografts in allochimeric-conditioned hosts exhibited moderate B cell infiltration when compared to rejecting controls. Analysis of intragraft cytokines revealed selective upregulation of IL-10 and marked inhibition of IL-2, IFN-γ, and IL-4. Our findings indicate the emergence of a peripherally induced tolerant state, afforded by the novel approach of soluble class I allochimeric conditioning that presents donor immunogenic epitopes in the context of recipient class I determinants.     

Evolutionary History of MHC Class I Genes in the Mammalian Order Perissodactyla

We carried out an analysis of partial sequences from expressed major histocompatibility complex (MHC) class I genes isolated from a range of equid species and more distantly related members of the mammalian order Perissodactyla. Phylogenetic analysis revealed a minimum of six groups, five of which contained genes and alleles that are found in equid species and one group specific to the rhinoceros. Four of the groups contained only one, or very few sequences, indicating the presence of relatively nonpolymorphic loci, while another group contained the majority of the equid sequences identified. These data suggest that a diversification of MHC genes took place after the split between the Equidae and the Rhinocerotidae yet before the speciation events within the genus Equus. Received: 17 November 1998 / Accepted: 7 April 1999     

A Genome-Wide Screen for Machinery Involved in Downregulation of MHC Class I by HIV-1 Nef

The HIV-1-encoded protein, Nef, plays a key role in the development of AIDS. One of Nef’s functions is to keep MHC class I off the surface of infected cells, a process that requires the host proteins clathrin and AP-1. To identify other proteins involved in this pathway, we carried out a genome-wide siRNA library screen on HeLa cells co-expressing HLA-A2 and an inducible form of Nef. Out of 21,121 siRNA pools, 100 were selected for further analysis, based on their ability to either inhibit or enhance downregulation of MHC-I by Nef. When cells were treated with the same siRNA pools as those used in the screen, 79% produced a similar phenotype. However, when the cells were treated with different siRNA reagents targeting the same genes, only 16% produced a similar phenotype. This indicates that most of the hits found in the original screen are likely to have been off-target, an important concern that is often not taken into account in siRNA screening studies. Nevertheless, we identified novel host factors involved in Nef-induced downregulation of MHC-I, including four genes, MIIP, CAMSAP3, SLC6A3, and KCTD19, where multiple reagents produced a strong inhibitory effect on Nef activity. Other hits slightly below our very high stringency cutoff point may also deserve further study. Thus, our dataset is a valuable resource for scientists investigating the pathogenesis of HIV.     

Activation by serotonin of starfish eggs expressing the rat serotonin 1c receptor.

Starfish oocytes were injected with mRNA for the serotonin 1c receptor or with rat brain poly A+ mRNA, incubated to allow expression of the membrane protein, then matured to eggs by addition of 1-methyladenine. Applying serotonin to these eggs caused cortical granule exocytosis like that occurring at fertilization. Because the serotonin 1c receptor specifically activates a G-protein, these results provide support for the hypothesis that sperm activate eggs by way of a receptor-G-protein interaction. The starfish oocyte may be a generally useful system for expression of exogenous mRNA for membrane proteins.     

Intragraft Selection of the T Cell Receptor Repertoire by Class I MHC Sequences in Tolerant Recipients

Background

Allograft tolerance of ACI (RT1^a) recipients to WF (RT1^u) hearts can be induced by allochimeric class I MHC molecules containing donor-type (RT1A^u) immunogenic epitopes displayed on recipient-type (RT1A^a) sequences. Here, we sought the mechanisms by which allochimeric sequences may affect responding T cells through T cell receptor (TCA) repertoire restriction.

Methodology/Principal Findings

The soluble [α_1h ^u]-RT1.A^a allochimeric molecule was delivered into ACI recipients of WF hearts in the presence of sub-therapeutic dose of cyclosporine (CsA). The TCR Vβ spectrotyping of the splenocytes and cardiac allografts showed that the Vβ gene families were differentially expressed within the TCR repertoire in allochimeric- or high-dose CsA-treated tolerant recipients at day +5 and +7 of post-transplantation. However, at day 30 of post-transplantation the allochimeric molecule-treated rats showed the restriction of TCR repertoire with altered dominant size peaks representing preferential clonal expansion of Vβ7, Vβ11, Vβ13, Vβ 14, and Vβ15 genes. Moreover, we found a positive correlation between the alteration of Vβ profile, restriction of TCR repertoire, and the establishment of allograft tolerance.

Conclusions

Our findings indicate that presentation of allochimeric MHC class I sequences that partially mimic donor and recipient epitopes may induce unique tolerant state by selecting alloresponsive Vβ genes.