Antigen Processing

RESEARCH ON ANTIGEN PROCESSING AND PRESENTATION USING THE HEL PROTEIN

 

These are some of our previous studies. We no longer do much research on HEL antigen processing and presentation as our focus has redirected toward studying insulin as a model antigen.

 

Presentation of HEL-early studies. We analyze the cellular and molecular events that transpire in APC when it takes up an antigenic protein, and that results in the generation of a complex of processed peptide and class II-MHC molecules, a pMHC complex. We use hen egg white lysozyme (HEL) as a model protein.

APC processes HEL into four major peptide families, identified immunologically as well as biochemically by analysis of the pMHC complex using mass spectrometry (MS). The chemical basis for their selection was studied by combinations of binding analysis using purified I-Ak molecules and mutagenesis of the peptides, HEL, or the I-Ak molecules. Quantitation of all families was accomplished by developing monoclonal anti-peptide antibodies for use in sensitive ELISAs of the extracted peptides, or as capture reagents for the MS studies. The same families of peptides were selected by the three sets of APC, i.e. dendritic cells (DC), macrophages, or B cells. The most abundantly selected peptides show an acidic residue corresponding to P1 plus a constellation of favorable residues at P4, P6 and P9. Peptide 48-62 of HEL (DGST DYGILQINS RWW - underlined is the 9-amino acid core sequence), was abundantly selected, even occupying as much as 10% of the class II bound peptides. A second set of peptides were selected on a much weaker binding motif, having asparagines at P4 as their major MHC contact residue with small polar residues at P1.

Knowing the density of each pMHC, the T cell repertoire to them was examined. A lack of correlation was noted between density of the various pMHC and the cellular CD4 T cell response. This is important information that tells us that a number of regulatory effects take place during presentations in vivo that influence the outcome.

A major component of all these studies has been the mass spectrometry analysis of peptides, which is done in the laboratory of Professor Michael Gross in the Chemistry Department of Washington University. The analysis includes the participation of Henry Rohrs and Manuel Plasencia.

Presentation of peptides generates unique pMHC complexes. We have identified two distinct sets of CD4 T cells to HEL as well as a number of proteins, including self-proteins. The conventional T cells - we use the term type A - respond to both protein and peptide in regular antigen presentation assays. The non-conventional T cells - called type B - respond only to peptides and not to the peptide generated from intracellular processing of the protein. The importance of the type B T cells is that those directed to autologous proteins escape thymic negative selection and can potentially participate in autoimmunity. We first identified the A/B T cells when studying presentation of HEL, but we subsequently found them to a number of autologous peptides. More importantly, we find them spontaneously in the NOD diabetic autoimmunity against insulin. See the Autoimmune Diabetes Research Program.

Two detailed reviews of the initial findings with HEL, done with Scott Lovitch who did his thesis work on this project, are: Conformational isomers of a peptide-class II MHC complex. Immunol Rev 2005; 207:293-313. PMID16181344; and The wide diversity and complexity of peptides bound to class II MHC molecules. Curr Opin Immunol 2006;18:70-77.

We describe briefly the initial findings. The first observations were based on the isolation of the peptides from HEL, presented by either I-Ek or I-Ak molecules. Although the major peptides, 84-96 or 48-62, respectively, were highly selected during processing and were easily isolated and identified by MS from MHC molecules, a number of T cells failed to recognize the processed peptide from HEL. These T cells only recognized the peptide offered to the T cells as an exogenous peptide, but not the peptide derived from the processing of HEL: we referred to them as type B T cells. (We refer to the type B pMHC complex and the corresponding T cells as type B T cells; we also refer to a free peptide that gives rise to type B pMHC as a "B-epitope".)

We ruled out a number of explanations: contaminants in the synthetic peptides used in the assays, post-translational modifications, or different binding registers. To note is that the type B T cells did not recognize a class II molecule with the peptide bound covalently. Importantly, the peptide extracted from the I-Ak molecule after processing of HEL - which was not recognized by the type B T cells - stimulated them, if isolated and offered exogenously. We concluded that the likely explanation was the mode of processing in the APC. Thus, HEL offered to APC generated only conventional type A T cells; peptide offered to APC generated, besides type A T cells, a second set, the type B, which was as abundant in number.

Central in our decision to study the type B T cells were the findings on their biology. We reasoned that if the finding was relevant we should find type B T cells in transgenic mice that express HEL in all their APC. These APC present the processed HEL peptides, but present weakly those pMHC derived from exogenous peptide. When HEL transgenic mice were immunized with HEL there was no response, as expected: a robust negative selection deleted all reactive T cells. However, immunization with peptide gave a different result. In normal mice, immunization with the 48-62 peptide gave rise to about an equal number of type A and B T cells. In contrast, in HEL transgenic mice, only the type B T cells were found, while all type A T cells were deleted.

The site of formation of the pMHC complex explains the generation of the two sets of unique pMHC complexes. Protein antigens traffic through vesicles to reach a late vesicular compartment, bearing class II molecules recently arrived from ER-Golgi. In this late vesicle, several steps are well known to take place: the protein antigen is reduced and partially catabolized, the generated peptide segments bind to the class II molecules as the CLIP peptide from invariant chain is released; H2-DM, a protein that is mostly restricted to late vesicles, interacts with the complex, releasing the weak or more unstable binding peptides. This interaction of the MHC molecules with DM results in the selection of the most stable pMHC, which then traffic to the cell surface. Importantly, in contrast to proteins, exogenous peptides do not reach late vesicles. Peptides (or denatured proteins) bind to class II MHC molecules by peptide exchange at plasma membrane or in recycling vesicle. Peptides are loaded in the absence of H2-DM, and thus unstable pMHC are not discriminated against. In brief, processing of the protein restricts the repertoire of pMHC generated from a given peptide segment, while peptides are free to generate from such a segment a diverse repertoire.

In the HEL system, distinct conformations of the peptide within the groove of the MHC molecule explains the type A/B pMHC complexes. The HEL 48-62 peptide complexed to I-Ak molecules exists in two states; one stable and long-lived that is not affected by editing by H2-DM molecules. A second state, short-lived and unstable, is negatively affected by interactions between H2-DM and the class II-MHC molecule. Beyond eliminating weak binding epitopes, H2-DM is also a conformational editor that eliminates the weak conformations. It follows that binding of free peptide to class II MHC molecules on the plasma membrane, or recycling vesicles allows for more flexibility of how the peptide sits in the binding groove.

Recently, type B pMHC complexes from HEL were generated from protein processing in a situation where the dendritic cells (DC) were activated by TLR ligands or interferon type 1. This interaction likely changed the traffic of the HEL molecule generating the pMHC in an H2DM free vesicle. This finding was replicated in vivo by immunizing mice with strong adjuvants and testing a T cell receptor transgenic mouse directed to a type B pMHC of HEL (MLA11.2). See below abstract from paper by Beverly Strong.

Post-translational modifications of peptides.
During antigen processing biochemical changes can take place on the peptides selected to bind to class II-MHC molecules. Peptides bearing post-translational modifications (PTM) can be immunogenic and generate T cells that only recognize the modified peptide. We found PTMs of HEL peptides bound to I-Ak molecules: specifically nitration of tyrosines, oxidation of tryptophans and arginine to citrulline changes, all of which induced specific CD4 T cells. The nitrated tyrosine peptides, or those peptides with oxidized tryptophans, were produced in APC activated by interferon-gamma that generated peroxynitrate, a strong oxidizing agent. Such T cells may be found after infections and may represent an important component of the anti-microbial repertoire.

Another important PTM involves the conversion of arginines to citrulline, a change catalyzed by peptidylarginine deiminases (PAD) enzymes, two of which, PAD-2 and PAD-4, are expressed in myeloid cells.. We identified citrullination of HEL peptides after processing of the protein, giving rise to highly specific CD4 T cells. [Cutting edge: T cells specifically recognize citrullinated peptides after immunization with protein antigens. J Immunol 2006;177:1421-1425.] This finding has to be placed in the perspective that patients with rheumatoid arthritis develop antibodies to citrullinated proteins; such antibodies serve as an excellent marker of disease. Our findings point out that the citrullination is not restricted to cells in the arthritic joint.

Recently, Jamie Rimer found that citrullination was tightly linked to an autophagy response in the APC, see below the abstract of her publication. We argue that there are two distinct processing sites in the APC, one that gives rise to the citrullinated peptides and which is part of an autophagosome; and one that gives rise to unmodified peptides, a conventional late processing vesicle. Jamie was able to biologically differentiate one from the other.

Presentation of class I-MHC epitopes from HEL.
Mice of the H2-g7 haplotype generate both a class II and a class I MHC epitope upon immunization with HEL. This has led us to examine and compare presentation of class I and II peptides in the APC. A summary of our initial evaluation can be found below in the abstract of the paper published in 2009 in PNAS. Roger Belizaire, who just finished his thesis work on this project, has identified conditions that influence presentation of each set of pMHC. HEL cross presentation is a vesicular event, it does not require the passage of HEL to the cytosol.

Presentation in vivo: blood-borne HEL in vivo.
We confirmed that the thymus was highly effective at taking up and presenting blood proteins such as HEL as a result of its DC network surrounding a restricted anatomical area. The permeability of blood proteins was largely dependent on molecular size and took place on a restricted anatomical area on the cortico-medullary zone. Thymic DC, both SIRPα positive and negative cDC subsets, and not TECs, were particularly effective at capturing and presenting blood-derived HEL. [The presentation of blood constituents may complement the function of the AIRE transcriptional regulator in presentation of tissue specific antigens. Indeed, some tissue-derived proteins and peptides are found in the circulation, although the extent of this representation still needs examination.]

We found a very narrow margin between the doses that resulted in the induction of negative selection (of both conventional CD single positive T cells and Tregs) and those that induced Tregs. Hence, the sensitivity of both negative selection and Treg induction to pMHC levels ensures that central tolerance will be effective, regardless of the constraints imposed by the levels of antigen expression. The exquisite efficiency, sensitivity and stringency of central tolerance took place not only by the presentation abilities of the DC system, but importantly by the low threshold of activation of the T cells during their thymic sojourn.

Presentation in vivo: role of adjuvants and neutrophils. Chiao-Wen Yang examined the effects of adjuvants on immune responses to known epitopes from protein antigens including HEL. Chiao-Wen found that neutrophils entered lymph nodes draining the immunization site wiyhin a few minutes after immunization. Neutrophils entered via lymphatics and were prominent in the cortical sinus. By depleting neutrophil using specific antibodies or by examining genetically neutropenic mice she found a marked suppressive effect of neutrophils on CD4 T cell, as well as antibody responses. Neutrophils modulated the extent of T cell response through several mechanisms, including competing for available antigen, but most critically by affecting the interaction between Dc and T cells by way of prostaglandins. In a second publicatoion on this subject  neutrophils were found  to be the major cells controlling the spread of T cell responses to distal lymph nodes. In the presence of neutrophils, ~75% of the response was restricted to the draining node,  but in their absence, most of the response was found in distal nodes. Prostanoids were responsible for the rapid entry of neutrophils into the draining nodes, as well as for the two distinct neutrophil effects: the modulation of the magnitude of the cellular response, and in its spread outside the draining nodes. Neutrophil-produced thromboxane A(2) was the key eicosanoid controlling both effects. Adoptive transfer of neutrophils into mice genetically deficient in neutrophils indicated their role in both. These functions of neutrophils are important in infections and vaccinations with adjuvants where neutrophils are abundant in the initial stages.

Below are abstracts of recent publications dealing with antigen presentation using the HEL protein

T cells distinguish MHC-peptide complexes formed in separate vesicles and edited by H2-DM. Zheng Pu Z, Scott Lovitch, Elizabeth Bikoff, Emil Unanue. Immunity 20:467-476, 2004.

The peptide spanning residues 48-61 of hen egg white lysozyme (HEL) presented by I-A(k) gives rise to two T cell populations, referred to as type A and B, that distinguish the complex generated intracellularly upon processing of HEL from that formed with exogenous peptide. Here, we ascribe this difference to recognition of distinct conformers of the complex and show that formation of the two complexes results from antigen processing in different intracellular compartments and is dependent upon H2-DM. While the type A complex preferentially formed in a lysosome-like late vesicle, the type B complex failed to form in this compartment; this distinction was abolished in antigen-presenting cells lacking DM. Experiments in vitro indicated that H2-DM acts directly on the complex to eliminate the type B conformation. We conclude that different antigen-processing pathways generate distinct MHC-peptide conformers, priming T cells with distinct specificity that may play unique roles in immunity.

Targeting proteins to distinct subcellular compartments reveals unique requirements for MHC class I and II presentation. Belizaire R, Unanue ER. Proc Natl Acad Sci USA 2009;106:17463-17468.


Peptides derived from exogenous proteins are presented by both MHC class I and II. Despite extensive study, the features of the endocytic pathway that mediate cross-presentation of exogenous antigens on MHC class I are not entirely understood and difficult to generalize to all proteins. Here we used dendritic cells (DC) and macrophages (MФ) to examine MHC class I and II presentation of hen egg-white lysozyme (HEL) in different forms, soluble and liposome-encapsulated. Soluble HEL or HEL targeted to a late endosomal compartment only allowed for MHC class II presentation, in a process that was blocked by chloroquine and a cathepsin S (CatS) inhibitor; brefeldin A (BFA) also blocked presentation, indicating a requirement for nascent MHC class II. In contrast, liposome-encapsulated HEL targeted to early endosomes entered the MHC class I and II presentation pathways. Cross-presentation of HEL in early endosomal liposomes had several unique features: it was markedly increased by BFA and by blockade of the proteasome or CatS activity; it occurred independently of the transporter associated with antigen processing (TAP) but required an MHC class I surface-stabilizing peptide; it was inhibited by chloroquine. Remarkably, chloroquine facilitated MHC class I cross-presentation of soluble HEL and HEL in late endosomal liposomes. Altogether, MHC class I and II presentation of HEL occurred through pathways having distinct molecular and proteolytic requirements. Moreover, MHC class I sampled antigenic peptides from various points along the endocytic route.

Thymus-blood protein interactions are highly effective in negative selection and regulatory T cell induction. Atibalentja DF, Byersdorfer CA, Unanue ER. J Immunol 2009;183:7909-7918.


Using hen egg-white lysozyme (HEL), the effect of blood proteins on CD4 thymic cells was examined. A small fraction of intravenously injected HEL rapidly entered the thymus into the medulla. There it was captured, and presented by dendritic cells (DCs) to thymocytes from two T-cell receptor transgenic mice, one directed to a dominant peptide, a second to a poorly displayed peptide, both presented by MHC class II molecules I-Ak. Presentation by DC led to negative selection and induction of regulatory T cells (Tregs), independent of epithelial cells. Presentation took place at very low levels, less than 100 peptide-MHC (pMHC) complexes per DC. Such low levels could induce negative selection, but even lower levels could induce Tregs. The anatomy of the thymus-blood barrier, the highly efficient presentation by DC, together with the high sensitivity of thymic T cells to pMHC complexes, results in blood protein antigens having a profound effect on thymic T cells.

Neutrophils influence the level of antigen presentation during the immune response to protein antigens in adjuvants. Yang C-W, Strong BSI, Miller MJ, Unanue ER. J Immunol 2010;185:2927-2934.

Neutrophils modulated Ag presentation following immunization with Ags in CFA or IFA or alum. The neutrophils had an important negative role in the CD4 T cell and B cell responses to three protein Ags: hen egg white lysozyme, OVA, and listeriolysin O. In their absence (by depleting with Abs for only the first 24 h, or using genetically neutropenic mice), the cellular responses increased several-fold. The CD8 response was not affected or slightly decreased. Competition for Ag between the presenting cells and the neutrophils, as well as an effect on the response to Ag-bearing dendritic cells (DCs), was documented. Neutrophils entered the draining lymph nodes rapidly and for a brief period of several hours, localizing mainly to the marginal sinus and superficial cortex. There they established brief contact with DCs and macrophages. Moreover, neutrophils imprinted on the quality of the subsequent DC-T cell interactions, despite no physical contact with them; by intravital microscopy, the clustering of Ag-specific T cells and DCs was improved in neutropenic mice. Thus, neutrophils are obligate cells that briefly enter sites of immunization and set the level of Ag presentation. A brief depletion may have a considerably positive impact on vaccination.

Functional redundancy between thymic CD8α+ and Sirpα+ conventional dendritic cells in presentation of blood-derived lysozyme by MHC class II proteins. Atibalentja DF, Murphy KM, Unanue ER. J Immunol 2011;186:1421-1431.

We evaluated the presentation of blood-derived protein Ags by APCs in the thymus. Two conventional dendritic cells (cDCs), the CD8α(+)Sirpα(-)CD11c(hi) (CD8α(+) cDC) and the CD8α(-)Sirpα(+)CD11c(hi) (Sirpα(+) cDC), were previously identified as presenting MHC class II bound peptides from hen egg white lysozyme (HEL) injected intravenously. All thymic APCs acquired the injected HEL, with the plasmacytoid dendritic cell being the best, followed by the Sirpα(+) cDC and the CD8α(+) cDC. Both cDCs induced to similar extent negative selection and regulatory T cells in HEL TCR transgenic mice, indicating a redundant role of the two cDC subsets in the presentation of blood-borne HEL. Immature dendritic cells or plasmacytoid dendritic cells were considerably less efficient. Batf3(-/-) mice, with significantly reduced numbers of CD8α(+) cDCs, were not impaired in HEL presentation by I-A(k) molecules of thymic APCs. Lastly, clodronate liposome treatment of TCR transgenic mice depleted blood APCs including Sirpα(+) cDCs without affecting the number of thymic APCs. In such treated mice, there was no effect on negative selection or regulatory T cells in mice when administering HEL, indicating that the T cell responses were mediated primarily by the cDCs localized in the thymus.

Presentation of type B peptide-MHC complexes from hen egg white lysozyme by TLR ligands and type 1 interferons independent of H2-DM regulation. Strong BSI, Unanue ER. J Immunol 2011;187:2103-2201.

In APCs, presentation by MHC II molecules of the chemically dominant peptide from the protein hen egg white lysozyme (HEL) generates different conformational isomers of the peptide-MHC II complexes (pMHC). Type B pMHCs are formed in early endosomes from exogenous peptides in the absence of H2-DM, whereas in contrast, type A pMHC complexes are formed from HEL protein in late vesicles after editing by H2-DM. Thus, H2-DM edits off the more unstable pMHC complexes, which are not presented from HEL. In this study, we show that type B pMHC complexes were presented from HEL protein only after stimulation of dendritic cells (DC) with TLR ligands or type I IFN. Type I IFN contributed to most TLR ligand-induced type B pMHC generation, as presentation decreased in DC lacking the receptor for type I IFNs (IFNAR1(-/-)). In contrast, presentation of type A pMHC from HEL and from peptide was minimally affected by TLR ligands. The relative effectiveness of CD8α(+) DC or CD8α(-) DC in presenting type B pMHC complexes varied depending on the TLR ligand used. The mechanisms of generation of type B pMHC from HEL protein with TLR stimulation did not involve H2-DM or release of peptides. DC from H2-DM-deficient mice in the presence of TLR ligands presented type B pMHC. Such DC showed a slight enhancement of HEL catabolism, but peptide release was not evident. Thus, TLR ligands and type I IFN alter the pathways of presentation by MHC II molecules of DC such that type B pMHCs are generated from protein Ag.

Autophagy in antigen presenting cells results in presentation of citrullinated peptides to CD4 T cells. Ireland JM, Unanue ER. J Exp Med 2011;208:2625-2632.

Antibody responses to citrullinated self-proteins are found in autoimmunities, particularly in rheumatoid arthritis, where they serve as a diagnostic indicator. We show here that processing of the protein hen egg-white lysozyme (HEL) resulted in citrullination of peptides presented on class II MHC molecules by antigen-presenting cells. The presentation of the citrullinated peptides but not of the unmodified peptides was associated with autophagy. Dendritic cells (DCs), macrophages, and thymic DCs presented citrullinated peptides constitutively. Their treatment with 3-methyladenine (3MA) blocked presentation of citrullinated HEL peptides, but presentation of unmodified peptides was not affected. Presentation of citrullinated peptides was not detected on B cells or B lymphoma cells under normal culture conditions. In B cells, engagement of the B cell antigen receptor was required for presentation of the citrullinated peptides, also inhibited by 3MA. B lymphoma-expressing HEL cells presented citrullinated peptides only after brief serum starvation. This presentation was reduced by 3MA or by reduction in Atg5 expression. Presentation of the unmodified peptides was not changed. The findings indicate a linkage between autophagy and autoreactivity through the generation of this neo-epitope.

Neutrophils control the magnitude and spread of the immune response in a thromboxane A2-mediated process. Yang CW, Unanue ER. J Exp Med. 2013 Feb 11;210(2):375-87.

Neutrophils are obligate cells entering lymph nodes shortly after immunization with protein antigens in adjuvants, starting during the first hour and continuing for several days in two distinct waves. Previously, we demonstrated the strong suppressive effects of neutrophils on CD4 T cell and B cell responses, using either neutrophil-depleting antibodies or genetically neutropenic mice. In this study, we find that neutrophils are the major cells controlling the spread of T cell responses to distal lymph nodes. Although in the presence of neutrophils, ~75% of the response was restricted to the draining node, in their absence, most of the response was found in distal nodes. Prostanoids were responsible for the rapid entry of neutrophils into the draining nodes, as well as for the two distinct neutrophil effects: the modulation of the magnitude of the cellular response, and in its spread outside the draining nodes. Neutrophil-produced thromboxane A(2) was the key eicosanoid controlling both effects. Adoptive transfer of neutrophils into mice genetically deficient in neutrophils indicated their role in both. These functions of neutrophils are important in infections and vaccinations with adjuvants where neutrophils are abundant in the initial stages.