The melting pot of the MHC II peptidome

Abstract

Recent advances in mass spectrometry technology have facilitated detailed examination of MHC-II immunopeptidomes, i.e. the repertoires of peptides bound to MHC-II molecules expressed in antigen presenting cells. These studies have deepened our view of MHC-II presentation. Other studies have broadened our view of pathways leading up to peptide loading. Here we review these recent studies in the context of earlier work on conventional and non-conventional MHC-II processing. The message that emerges is that sources of antigen beyond conventional endosomal processing of endocytosed proteins are important for generation of cellular immune responses to pathogens and maintenance of central and peripheral tolerance. The multiplicity of pathways results in a broad MHC II immunopeptidome that conveys the sampled environment to patrolling T cells.

Presentation of endogenous and exogenous antigens

From the very first characterizations of eluted peptides [1–3], MHC II molecules have been shown to sample both endogenous and exogenous antigens. The inclusion into routine mass spectrometry workflows of once-esoteric techniques such as ion trapping, electron-transfer dissociation, ion-mobility fractionation, and data-independent acquisition strategies has allowed several groups to characterize very large sets of naturally-processed peptides bound to MHC II molecules [4–7]. Such sets can comprise several thousands of individual peptides derived from hundreds of different proteins, and now rival (or even exceed) in terms of numbers of confidently sequenced eluted peptides the most detailed studies of MHC I molecules [8–10]. By combining several analysis strategies, Mommen et al were able to identify almost 14,000 peptides bound to three HLA-DR allotypes expressed by a myelo-monocytic leukemia cell line [4]. The peptides derived from almost 2000 different source proteins, and appear to represent the most complete MHC II peptidome to date. Bergseng et al characterized up to 7400 HLA-DQ-bound peptides from each of several EBV-transformed B-lymphoblastoid cell lines, and used these to define peptide binding motifs for three celiac disease-associated DQ allotypes [5]. Similarly Sofron et al identified over 1000 peptides from the murine A20 lymphoma line, helping to refine the I-A^d motif [6]. In each of these studies, the majority of eluted peptides derived from endogenous proteins, with a substantial fraction (up to 20%) derived from exogenous proteins brought into the cell by endocytosis. Of the endogenous sources of peptides, membrane proteins from plasma membrane or endo/lysosomal compartments were well represented, but so were nuclear, mitochondrial, ER/golgi, and cytosolic compartments.

Some studies characterized immunopeptidomes of antigen presenting cells as isolated directly from tissues. These should more closely reflect the distribution of source proteins and in vivo peptide repertoire than do the cell lines studies described above. Sofron et al reported almost 3000 I-A^d binding peptides from BALB/c spleen, and a similar number of I-A^b-binding peptides from C57BL/6 spleen [6]. As in the cell line studies described above, and as described previously for I-A^b from murine splenocytes [11], peptides bound to MHC II in vitro derive from proteins present throughout the cell. Collado et al identified 247 different peptides in three samples of human thymus [12], extending a much earlier study of mouse thymus that showed generally similar sets of self-peptides presented in thymus and spleen [13]. A subsequent study from the same group identified, among the peptides presented in human thymus, two peptides derived from AIRE-dependent expression of prostate and axon specific antigens in medullary epithelial cells [14], directly confirming the role of AIRE in providing antigens for presentation by MHC II molecules as part of central tolerance mechanisms. Clement et al identified more than 3000 MHCII-bound peptides presented in vivo on dendritic cells from HLA-DR1-transgenic mice [7]. Among the ~10% of peptides identified in this study that derived from extracellular protein sources, several corresponded to peptides that were also identified in lymphatic fluid where they had been generated by extracellular tissue remodeling, cellular apoptosis, plasma membrane editing and complement/coagulation protease cascade.

Lymph as a source of antigen for MHCII presentation

Lymph derives from the extracellular fluid bathing all tissues in the body. It collects the products of metabolic and catabolic activities underway in parenchymal organs [15] and so it provides a snap shot of the cellular and extracellular proteome in physiological and pathological conditions. In addition to intact antigens, lymph contains many peptides, generated by extracellular matrix reprocessing, cell-surface receptor editing, and other processes including release of material generated by cell death pathways [16]. Clement et al compared peptides present in lymph and peptides eluted from conventional (splenic) dendritic cells, and in many cases found similar species, often with termini that matched known sites of extracellular processing [7]. For example, a peptide derived from complement 3 was found in identical form both in lymph and bound to HLA-DR1 presented on splenic dendritic cells. The source protein is present in many tissues including spleen but is not expressed in dendritic cells; moreover its processing is known to require complement 1 as part of the complement activation pathway. In vitro processing studies showed that the observed peptide termini were not generated by digestion of purified C3 with isolated endosomes or mixtures of cathepsins, although cleavage at many other sites was observed [7]. These observations indicated that the dendritic cells were able to capture and present a pre-processed version of the epitope. Several other such peptides were identified, from gelsolin, thymosin β4, and collagen II [7]. Overall lymph-carried peptides were shown to be generated by a variety of processing pathways including cathepsins, caspases, granzymes, and matrix metalloproteases among others. In many cases the eluted peptides were highly sensitive to DM-mediated peptide exchange, supporting the idea that the pathway by which they were loaded and presented did not include DM-containing endosomes. One potential mechanism for loading lymph-derived peptides onto MHCII proteins could involve the cell surface [17] or shallow recycling compartments [18–20] (Figure 1). MHC II loading could be achieved through exchange with low affinity/low stability peptides or on empty MHC II molecules that are expressed on the surface of immature dendritic cells [21,22]. Peptide-free MHCII proteins convert between peptide-receptive and peptide-averse conformations [23–26], and receptive MHCII could readily bind peptide provided extracellularly [22]. Interestingly, certain class I MHC proteins are also relatively stable as peptide-free species [27], and it has been shown that APC expressing these proteins also are able to directly access soluble extracellular antigen [28].

Fig 1.

Intracellular sites for conventional and non-conventional loading onto MHC II proteins. Extracellular antigens or cell-surface proteins can enter the MHC II pathway through a conventional route of endocytosis followed by proteolysis and MHC II loading in late endosomes or lysosomes. Alternate pathways for antigen entry include macroautophagy (MA), endosomal microautophagy (eMI), or chaperone-mediated autophagy (CMI), which bring proteins into late endosomes or lysosomes for processing and loading. Antigens can be loaded onto MHC II proteins also at the cell surface or in recycling, sorting, or early endosomes. Because of lower proteolytic activity in these compartments this pathway is likely to be more important for preprocessed peptides, for example from lymph, or for unfolded proteins. DM activity increases with endosomal maturation, in part because of DO dissociation from DM at low pH [107], and so DM-sensitive antigens are more likely to be loaded in early compartments, and loading in late compartments more likely to be restricted to DM-resistant antigens.

MHC II loading of exogenous peptides delivered intrathymically [29], intravenously [30], or intraperitoneally [31] have been shown to be effective ways to achieve central and peripheral tolerance [30–39]. Such peptides likely are conveyed to dendritic cells via lymph. The role of migratory DC in transporting the injected peptides to the thymus to mediate negative selection [35] and to lymph nodes to mediate peripheral T cell anergy and Treg induction [36,40] also have been reported [35,41]. However, extracellularly processed peptides also can also be a target for autoimmune responses [42]. The balance between tolerance and autoimmunity likely is determined by differences in peptide MHC II binding affinity/stability, efficiency of thymic selection, Treg induction, peptide generation by de novo processing pathways, and nature of the presenting APC. It is likely that several different processing pathways are utilized to generate the full variety of self-peptides required to maintain effective central and peripheral tolerance.

Autophagy and the MHCII peptidome

Many peptides identified in MHCII peptidomes derive from proteins found outside the classic endosomal/lysosomal pathway. Indeed, peptides derived from nuclear and cytosolic proteins together comprised 25% to 55% of the total HLA-DR-bound peptide sequences identified in two recent high-resolution studies [4,7]. It is likely that these antigens intersected the MHCII antigen processing pathway via an autophagy pathway. Three forms of autophagy have been described in immune cells, macroautophagy, microautophagy, and chaperone-mediated autophagy [43]. Macroautophagy is a “bulk” form of transport characterized by the sequestration of cargo from the cytosol into double membrane vesicles called autophagosomes, which fuse with late endosomes (to generate amphisomes) or lysosomes (to generate autophagolysosomes) [43,44]. Through this pathway cytosolic organelles are disposed, mitochondria (mitophagy), ribosomes (ribophagy), protein aggregates (aggregophagy), as well as sequestered cytosolic proteins [45,46]. Whereas in most cell types macroautophagy is induced, notably by starvation, in DC there is a constitutive macrophautophagic flux to deliver cytosolic self-proteins for immunosurveillance [47]. Pathogens also can be delivered to MHC II compartments through macroautophagy when the pathogens are present in the cytosol or when the autophagy machinery aids the fusion of the phagosome with endo-lysosomal compartments [48–50]. Macroautophagy-deficient mice have defective immunity to mycobacteria and decreased response to yellow fever virus and BCG vaccination [51–54]. Microautophagy derives from the inward invagination and pinching off of the late endosomal or lysosomal limiting membrane. In late endosomes, the process is ESCRT-mediated and the resulting vesicles contain soluble cytosolic proteins. The same vesicles can then be released as exosomes or degraded and release their cargo into the lumen of the late endosomal compartment [55]. Chaperone-mediated autophagy relies on hsc-70 recognition of selected cargo proteins (KFERQ-like motives) which are delivered to lysosomes through the LAMP-2A translocation complex [56]. Interestingly all forms of autophagy rely on the hsc-70 chaperone to transport cytosolic peptides, misfolded polypeptides, or ubiquitinated proteins into MHC II-containing organelles [43,57]. In DC a decrease in endosomal transport of cytosolic self-antigens has been observed upon knock down of macroautophagy and ESCRT-mediated microautophagy [55], in thymic epithelial cells macroautophagy has been shown to shape the T cell repertoire [58], and in a B cell line knock down of chaperone-mediated autophagy decreased MHC II presentation of a cytosolic protein [59]. Overall the three forms of autophagy are the major pathway by which cytosolic and organelle-derived self-proteins as well as certain pathogens are delivered to MHC II compartments [48].

Endosomal and non-endosomal processing pathways

Many studies over the last two decades have highlighted the role of non-canonical processing in the MHCII pathway. MHCII can acquire peptide in intracellular compartments other than the classical late endosomes and lysosomes rich in cathepsins and DM. Early work from several groups identified an early endosomal/plasma membrane compartment where peptide loading occurs on recycled MHC II molecules [20,28,60–62]. Processing in different compartments led to presentation of different epitopes [62–64]. In early endosomes, invariant chain could be removed from MHC II and exachanged for peptides or unfolded proteins in a cathepsin-independent, DM-independent manner [20]. Capture of peptides and proteins at the plasma membrane also has been reported [65–67]. The feasibility of a capture-first model for antigen processing was highlighted by recent work from Sadegh-Nasseri and Kim in their development of a mimimalist in vitro antigen processing system that coupled proteolysis and loading steps [68,69].

An important role for non-endosomal processing in generation of the MHC II immunopeptidome was emphasized by recent work by Bosch et al, in studies of dendritic cells with disrupted multivesicular bodies which nonetheless were able to process and load peptides for MHC II presentation via an early endosome or cell surface peptide loading pathway [70]. For at least some antigens, components of the MHC I processing pathway can be involved. Spencer et al showed recently that the profile of peptides eluted from I-A^b was substantially altered in splenocytes from TAP−/− or ERAAP−/− mice, with effects on CD4 T cell recognition of epitopes derived from Listeria or vaccinia virus infection [71]. Tewari et al earlier had identified E^d restricted influenza epitopes similarly dependent on proteasome and TAP-mediated processing [72]. How antigens processed by the MHC I pathway intersect MHC II proteins is not clear, but processing machinery from both pathways can be found together in some cell types [73–75]. Altogether these studies delineate how diverse processing mechanisms in conventional late endosomes and elsewhere ensure that a broad peptidome representing many intracellular and extracellular proteins is presented by MHCII proteins.

DM-dependent and DM-independent loading

The ability of MHCII to load peptides in relatively shallow recycling compartments or at the cell surface suggests that loading can occur with little or no contribution from DM. Of course, many in vitro studies have shown that DM can stabilize peptide-free MHCII molecules and promote peptide exchange [76–83]. Recently, Yin et al reexamined this issue, and found that DM’s role in stabilizing peptide-free HLA-DR was facilitative rather than obligatory [84]. Both the rate of conversion between receptive and averse conformations, and the rate of peptide loading to receptive molecules, were increased by DM, but these processes were observed to occur efficiently also in the absence of DM. In vitro peptide exchange clearly is facilitated by DM, but with different rate enhancements for different peptides [85–92]. Clement et al reported DM susceptibilities for some abundant peptides eluted from splenic DC, as part of their study on the contribution of lymph peptides to the MHC II peptidome [7]. A substantial fraction of the eluted peptides had rapid exchange rates even in the absence of DM. Thus some MHCII molecules at the surface or in shallow recycling compartments should be able to bind peptide even if DM were absent or inhibited by DO. In fact, mice lacking DM are capable of loading exogenously added peptides despite allele-dependent increases in CLIP levels and compromised ability to process intact antigen [93–95], The original phenotype described for DM-deficient cells was alteration in the ability to present native exogenous protein antigens but peptides derived from these same proteins were presented effectively [96]. Even in DM-sufficient mice, removal of CLIP and peptide loading in recycling or early endosomes can occur independently of DM [20,62]. In dendritic cells, processing in DM-dependent or DM-independent pathways can be regulated by TLR ligands and type I interferon [97,98]. Different epitopes are targeted in DM-deficient and DM-sufficient mice [64,99,100], although DM can be required for pathogen clearance [101]. Thus, MHC II peptide loading can occur in many scenarios in the absence of DM, and a lack of DM expression should not be considered to rule out MHC II presentation. An important role for DM-independent antigen processing in the antiviral response was demonstrated recently by Miller et al, who showed that a majority of I-Ab restricted epitopes in the C57BL/6 response to influenza were processed by non-canonical pathways including those not dependent on DM or invariant chain, with considerable diversity in the pathways accessed by particular epitopes [102]. Overall, in several scenarios, DM-independent loading can broaden the MHC II peptidomes presented at the cell surface.

Concluding Remarks

Peptides destined for loading onto MHC II proteins are generated by both endosomal and non-endosomal processing, and derive from proteins present in a wide variety of intracellular and extracellular compartments. Peptides produced by non-endosomal processing have been shown to have key roles in maintenance of peripheral tolerance and immunity to pathogens. Continuing work is needed to elucidate how the MHCII peptidomes changes during inflammatory conditions. Many peptides identified in the MHCII immunopeptidome studies carry post-translational modifications such as acetylation, amidation/deamidation, carboxylation, citrullation, hydroxylation/dehydration, methylation/demethylation, oxidation, and phosphorylation [7,9,103,104]. How these modifications impact tolerance and immune recognition in many cases remains to be determined. Because of the increased role of DM in MHC II processing under inflammatory conditions, the abundance of many self-peptides displayed at the cell surface is likely to change, with implications for the regulation of self-tolerance. Methodology for the reliable quantitative measurement of peptide abundances in complex samples is available, and very recently has begun to be applied to immunopeptidomes [105,106]. Thus we hope to learn in even greater detail about the distribution of peptides in the MHC II melting pot, and how they regulate T cell activity.

Highlights.

• MHCII present antigens from a variety of intracellular and extracellular sources

• Endosomal and non-endosomal processing pathways contribute to the MHCII peptidome

• Lymphatic fluid is a rich source of pre-processed antigens for MHCII presentation

• Multiple processing pathways promote presentation of a broad repertoire of peptides

• Presentation of a broad peptide repertoire might be important for tolerance