The elucidation of non-classical MHC class II antigen processing through the study of viral antigens

Abstract

By convention, CD4⁺ T cells are activated predominantly by Major Histocompatibility Complex class II-bound peptides derived from extracellular (exogenous) antigens. It has been known for decades that alternative sources of antigen, particularly those synthesized within the antigen-presenting cell, can also supply peptides but the impact on T_CD4+ responses, sometimes considerable, has only recently become appreciated. This review focuses on the contributions that studies of viral antigen have made to this shift in perspective, concluding with discussions of relevance to rational vaccine design, autoimmunity and cancer immunotherapy.

Introduction

CD4+ T cells (T_CD4+) are key components in the adaptive immune responses to viral infections, receiving activation signals from antigen-derived peptides displayed at cell surfaces by major histocompatibility complex class II molecules (MHC-II). In elaborating a panoply of cytokines, T_CD4+ help B cells differentiate into antibody producing plasma cells and enhance the functions of MHC class I (MHC-I)-restricted cytolytic CD8⁺ T cells (T_CD8+) [1–4]. T_CD4+, like T_CD8+, can be cytolytic, an important capability in defense against several viruses including Epstein Barr virus (EBV) [5–7], ectromelia [8], influenza [9] and lymphocytic choriomeningitis virus [10]. Moreover, perforin⁺ T_CD4+ are prevalent in the blood of human immunodeficiency virus (HIV) and EBV infected patients [11].

‘Professional’ antigen-presenting cells (pAPCs) — dendritic cells (DCs), macrophages and B cells — constitutively express MHC-II, allowing them to play many key and unique roles in host defense. Under inflammatory conditions, non-professional APCs can be induced to express MHC-II and serve as APCs [12,13].

By convention [14,15], the production of MHC-II-bound peptides begins with internalization of extracellular (exogenous) proteins by APCs [16] that are subsequently cleaved into peptides by acid proteases in the endosomal/lysosomal compartments. Simultaneously, newly synthesized MHC-II molecules in the endoplasmic reticulum (ER) are directed to the endosomal compartment by the non-covalently associated chaperone invariant chain (Ii). Upon reaching the late endosome Ii undergoes degradation, with only the portion termed Class II associated invariant chain peptide (CLIP) [17,18] surviving as it occupies the peptide-binding groove, which provides protection from proteolysis [19]. Processed peptides are exchanged for CLIP in a reaction that is catalyzed by H2-M (mouse) [20] or HLA-DM (humans) [21,22] heterodimer (hereafter referred to as DM).

Suggestions that there might be alternatives to this classical pathway were first reported over three decades ago in experiments that demonstrated the presentation of ‘self’ immunoglobulin proteins synthesized within the APC (‘endogenous’ processing) [23–26]. Subsequently, peptide elution and identification by mass spectrometry revealed that a large proportion of MHC-II-bound peptides are derived from cytosolic proteins [27–29]. Interspersed with these studies and from that time forward virus-based systems have substantially contributed to this issue in both reinforcing the notion of alternative MHC-II antigen processing and in providing mechanistic insights.

Alternative pathways involved in the generation of viral epitopes

Alternative exogenous processing — recycling MHC-II

An early study by Pinet et al. reported on an HLA-DR-restricted influenza hemagglutinin (HA) epitope whose presentation from input (exogenous) virus did not require Ii or MHC-II synthesis [30•]. Furthermore, truncating the cytoplasmic tails of DR, which prevented internalization, abrogated presentation [31]. In a subsequent publication the same group showed that the kinetics of presentation for this epitope were more rapid than another epitope processed by the classical pathway and required cysteine but not aspartic proteases (active in very low pH) evident from endosomal protease inhibitor experiments. Moreover, treatment with concanamycin B had no effect on its presentation but blocked presentation of the classically processed epitope, which suggested that presentation could take place in an early endocytic (EE) compartment [32]. Similar observations were made for a measles derived epitope, showing in addition that presentation is independent of DM expression [33]. While DM independence is generally the rule, DM dependence was described for an albumin derived peptide presented by recycling MHC-II [34].

We previously described an E^d-restricted peptide located within the stalk region of the influenza A/PR/8/34 HA that is, by all measures, presented by recycling MHC-II [35,36]. This is consistent with the biology of HA. Following internalization of virions, the stalk region of HA undergoes extensive unfolding in response to endosomal acidification, which triggers fusion of the viral and endosomal membranes and delivery of the viral genome to the cytosol. Since the class II peptide binding groove is open-ended the unfolded HA could ostensibly bind to recycling class II without any processing beyond separation from the virion. The extent to which recycling MHC-II contributes to the overall T_CD4+ response to any complex pathogen has not yet been determined as the cellular mechanisms that underpin recycling are still being elucidated [37].

Endogenous processing — autophagy

Many studies have associated endogenous MHCII presentation of intracellular proteins with autophagy, a catabolic process where cytoplasmic elements are delivered to endolysosomal organelles.

Macroautophagy involves the engulfment of cytosolic materials into double membrane vesicles called autophagosomes which then fuse with lysosomes to deliver its cargo for further degradation by lysosomal enzymes [38,39]. Work from the Munz laboratory, utilizing both a small molecule inhibitor, 3-methyladenine, and short interfering RNAs demonstrated that recombinant vaccinia virus produced (endogenous) Epstein-Barr Nuclear Antigen-1 (EBNA1) activates T_CD4+ via macroautophagy [5,40]. Of note, the presentation of three other epitopes within EBNA1 was substantially enhanced, in an autophagy dependent manner, upon ablation of the nuclear localization sequence, leading to the conclusion that location of the antigen governs whether macroautophagy is a viable processing mechanism [41]. In a Herpes Simplex Virus-1 mouse model, conditional knockout of the atg5 gene in conventional DCs, led to impaired T_CD4+ responses in vivo [42]. Recently, autophagy-mediated presentation of ovalbumin (OVA) expressed by recombinant modified vaccinia ankara virus (MVA) was observed in an in vitro murine bone marrow derived DC (BMDC) model [43••]. Of note, Blanchet et al. reported that DCs provided with inactivated HIV virions (exogenous source) can produce the gag1 epitope from gag p24 via autophagy [44], indicating that autophagy-dependent MHC-II presentation is not restricted to endogenous antigens. Autophagy may not play a role in the endogenous processing of some viruses. Atg7 knockdown had no impact on the global MHC-II presentation of influenza epitopes [45••]. Additionally, it has been recently shown that endogenous processing of HIV gag2 and env proteins by monocyte derived DCs or HeLa cells transduced with class II associated transcriptional activator (CIITA) proceeds by an autophagy independent mechanism [46••].

In chaperone-mediated autophagy (CMA), proteins containing the relatively degenerate pentapeptide KFERQ motif are transferred from the cytoplasm into lysosomes in a lysosome associated membrane protein-2a (LAMP-2a)-dependent manner [47]. CMA has been shown to be important for the presentation of cytosolic autoantigens such as glutamate decarboxylase and a modified form of human immunoglobulin K chain [48]. In the case of microautophagy, microvesicular like bodies are produced through invaginations on lysosomal membranes that envelop cytosolic proteins and organelles [49]. To our knowledge, CMA has not yet been implicated in the generation of virus-derived epitopes and microautophagy has not been implicated in the MHC-II processing of any antigen.

Endogenous processing — bona fide cytosolic processing

Autophagy could be viewed as a variant on the classical scheme as antigen processing still takes place in the endosomal compartment. There are, however, many instances of endogenous processing where cytosolic machinery is involved. First indications of this were in the late 1980s, when many groups reported that endogenously expressed viral proteins were presented to T_CD4+ [50–54].

The main engine for degradation of cytosolic proteins is the multicatalytic proteasome [55], which is best known for generating peptides conveyed via the transporter associated with antigen processing (TAP) into the endoplasmic reticulum for loading onto nascent MHC-I molecules and subsequent recognition by T_CD8+. However, the proteasome can also be instrumental for the generation of MHC-II-restricted epitopes. We have demonstrated with several epitopes that in vitro presentation from endogenous sources of influenza proteins is essentially abrogated when highly specific proteasome inhibitor is provided [56,57••]. What is more, proteasome-dependent epitopes appear to drive a substantial portion of the T_CD4+ response to influenza, on the order of 40–50% in the systems where analyses were conducted [56,57••]. Furthermore, we have reported that the vast majority of the T_CD4+ response to influenza, much larger than 50%, is driven by endogenous sources of antigen [57••]. Our failure to implicate macroautophagy in the processing of influenza antigens [45••] implies the existence of yet unidentified endogenous processing machinery.

Earliest indications that TAP can play a role in MHC-II processing were reported by the Long laboratory who tracked presentation of a minigene-encoded peptide derived from influenza HA and expressed by a recombinant vaccinia virus. Presentation was substantially reduced in the absence of TAP [58]. The processing pathway differed from that of classical MHC-I in being inhibited by chloroquine, a lysosomotropic agent, implying involvement of the endosomal compartment in antigen processing and/or peptide loading [59,60]. Later, studies from our lab reported that endogenous production of epitopes (requiring delivery of antigen to the cytosol) from full-length forms of the influenza neuraminidase (NA) and HA glycoproteins require both proteasome and TAP function [56]. The involvement of TAP does appear to be exceptional. TAP function was dispensable for HLA-DR1-restricted presentation of a cytosolically targeted HA [58]. Moreover, our mapping of C57Bl/6 T_CD4+ responses to live influenza led to the identification of thirteen I-A^b-restricted epitopes, all of which could be endogenously generated, and none requiring TAP [57••]. Of note, two of these epitopes, within the influenza nucleoprotein (NP), demonstrated full and partial proteasome dependence. Of even greater note, an NA-derived epitope was found to be dependent upon both gamma-interferon-inducible lysosomal thiol reductase (GILT) and proteasome function. Involvement of GILT is consistent with the epitope containing a cysteine residue that is disulfide bonded in the mature protein [61]. The mechanism by which a post-proteasomal intermediate is translocated into the EE, independent of TAP, for additional processing by GILT is currently under investigation. More recently, MVA-expressed OVA in BMDCs was also presented by MHC-II in a TAP-independent, proteasome dependent manner [43••].

The role for DM in loading of endogenously produced peptides appears to be quite variable. Dani et al. reported that the cytosolically targeted, proteasome dependent fusion protein I-Eα required DM for efficient loading on MHC-II [62]. On the other hand, data from our lab showed that presentation of the proteasome-, TAP-dependent, E^d-restricted epitopes within HA and NA do not require DM [56]. More recently we demonstrated with DM-knockout mice that nearly half of the thirteen IA^b-restricted influenza epitopes that we mapped can be presented in a DM independent manner [57••].

Conclusion

We have proposed that the highly diverse alternative MHC-II processing pathways described in brief here (Figure 1) substantially increase the array of peptides that can be presented, consequently enhancing T_CD4+ help to B cells and T_CD8+ [57••]. This is consistent with our observation that immunization of mice with low doses of live influenza (enabling endogenous processing) vs. much higher doses of inactivated virus (restricting processing to exogenous pathways) results in higher titers of antibody that are more protective [57••], and reminiscent of the repeated observation that a live influenza vaccine is more protective than an inactivated vaccine in inexperienced (pediatric) individuals [63]. Such results suggest that the notion of maximizing access to alternative MHC-II processing pathways should deserve strong consideration in rational vaccine design.

Figure 1. The Elucidation of Non-Classical MHC Class II Antigen Processing Through the Study of Viral A. Various alternative MHC-II processing pathways that have been described.

1. DM-independent recycling pathway [30•,31,32,33,35,36]; 2. Macroautophagy [5,40,42,43••]; 3. CMA [47,48]; 4a & 4b. Proteasome and TAP dependent pathway [58,56]; 5a & 5b. Proteasome dependent and TAP independent pathway [57••,58]; 6. Proteasome dependent, TAP independent and DM dependent pathway [62]. LE, late endosome; ERAD, ER associated degradation protein.

This review has focused on viral antigens but the same principles apply to the peptides that induce autoimmunity. As mentioned, targets of autoimmunity can be generated by chaperone-mediated autophagy [48] but endogenous processing has been reported for various autoantigens [64–68] and tumor antigens [69•,70]. If, as in the case of influenza, the majority of MHC-II-restricted self-peptides are produced by endogenous processing, systems that restrict candidate autoantigens to endosomal processing may miss opportunities for mechanistic insights and therapeutic strategies.

The importance of T_CD4+ in cancer immunotherapy is becoming increasingly apparent [71,72]. As successful cancer immunotherapy is essentially controlled autoimmunity, principles derived in the study of autoantigen processing might be successfully transferred to this arena.

Much remains to be learned about the alternatives to conventional MHC-II antigen processing. Increased investigation in this regard could provide important insights into how host responses to viral infections, self-antigens and tumor-specific antigens evolve, and how enhanced therapeutic approaches to all three areas might be developed.