Antigen Processing


Our previous studies examined antigen processing and presentation of hen egg white lysozyme (HEL) as a model antigen. As noted our recent studies on antigen processing and presentation has been redirected  toward studying insulin in autoimmune diabetes. In the perspective of our recent investigation in T1D we summarize the studies that led to the findings of two sets of CD4 T directed to a similar peptide segment of HEL.

We identified two distinct sets of CD4 T cells to HEL as well as to a number of proteins, including self-proteins. The conventional T cells - we use the term type A - responded to both protein and peptide in regular antigen presentation assays. The non-conventional T cells - called type B - responded 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 escaped 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 as detailed above.

The site of formation of the pMHC complex explained 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 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 traffics 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. 

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 presented the processed HEL peptides, but presented 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. In contrast, in HEL transgenic mice, only the type B T cells were found, while all type A T cells were deleted.

Viner NJ, Nelson CA, Deck B, Unanue ER:  Complexes generated by the binding of free peptides to class II MHC molecules are antigenically diverse compared with those generated by intracellular processing.  J Immunol 1996;156:2365-2368. PMID8786292 We investigated the specificity of T cell hybridomas isolated from mice immunized with synthetic peptides identical in sequence with the dominant, naturally processed, I-Ak-restricted peptides of hen egg lysozyme (HEL). Surprisingly, the majority of hybridomas showed little or no recognition of intact HEL after processing by different APCs. This was not an artifact caused by the use of synthetic peptides since the peptide-specific hybridomas responded to a tryptic digest of HEL or to naturally processed HEL peptides extracted from I-Ak. Thus, the interaction of free peptides with class II MHC molecules can generate complexes that are antigenically dissimilar to those resulting from intracellular processing of intact Ag. This has important implications both for the interpretation of experimental studies that involve peptide immunization and for the efficacy of peptide vaccination as a strategy for intervention in human disease.

Peterson DA, DiPaolo RJ, Kanagawa O, Unanue ER:  Quantitative analysis of the T cell repertoire that escapes negative selection.  Immunity 1999;11:453-462. PMID10549627

Peterson DA, DiPaolo RJ, Kanagawa O, Unanue ER:  Cutting edge:  A single MHC anchor residue alters the conformation of a peptide-MHC complex inducing T cells that survive negative selection.  J Immunol 2001;166:5874-5877. PMID11342600

Pu Z, Carrero JA, Unanue ER:  Distinct recognition by two subsets of T cells of an MHC class II-peptide complex.  Proc Natl Acad Sci USA 2002; 99:8844-8849. PMID12084929  We examine here the nature of the differential recognition by CD4+ T cells of a single peptide from hen-egg white lysozyme (HEL) presented by I-A(k) class II MHC molecules. Two subsets of T cells (called A and B) interact with the same peptide, each in unique ways that reflect the nature of the complex of peptide and MHC. We show that the A and B set of T cells can be distinguished by their functional interaction with the three T cell receptor (TCR) contact residues of the bound peptide. The dominant peptide of HEL selected from processing is bound in a single register where a critical TCR contact residue is situated about the middle of the core segment of the peptide: all T cells establish functional contact with it. Three sets of T cells, however, can be distinguished by their differential recognition of two TCR contacts situated at the amino and carboxyl sides of the central TCR contact residue. Type A T cells, the conventional cells that see the peptide after processing of HEL, need to recognize all three TCR contact residues. In contrast, the type B T cells recognize the peptide given exogenously, but not when processed: these T cells recognize either one of the peripheral TCR contact residues, indicating a much more flexible interaction of peptide with I-A(k) molecules. We discuss the mode of generation of the various T cells and their biological relevance.

Lovitch SB, Walters JJ, Gross ML, Unanue ER:  Antigen presenting cells present Abk-derived peptides which are autoantigenic to type B T cells.  J Immunol 2003;170:4155-4160. PMID12682247

T cells distinguish MHC-peptide complexes formed in separate vesicles and edited by H2-DM. 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.

Lovitch SB, Pu Z, Unanue ER:  Amino-terminal flanking residues determine the conformation of a peptide-class II MHC complex.  J Immunol 2006;176:2958-2968. PMID16493054

Lovitch SB, Unanue ER:  Conformational isomers of a peptide-class II MHC complex. Immunol Rev 2005; 207:293-313. PMID1618134

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 antigen-presenting cells (APC), presentation by class II-MHC 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, while 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. Here 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 interferons (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 class II MHC molecules of DC such that type B pMHCs are generated from protein antigen.
Autophagy in antigen presenting cells results in presentation

Other studies to note:

Herzog J, Maekawa Y, Cirrito TP, Illian BS, Unanue ER:  Activated antigen presenting cells select and present chemically modified peptides recognized by unique CD4 T cells.  Proc Natl Acad Sci USA 2005; 102:7928-7933. PMC1130168 

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.

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.

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.

Ireland JM, Unanue ER:  Autophagy in antigen presenting cells results in presentation of citrullinated peptides to CD4 T cells.  J Exp Med 2011;208:2625-2632.  PMC3244027

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.