Introduction
Antigen processing and loading of peptides onto MHC class II molecules is a multistep
process that involves vesicular transport of the MHCII molecules along the secretory
pathway, where they eventually merge with antigen-containing endocytic vesicles or
phagosomes (1). It is within these late endosomal or lysosomal compartments that protein
antigens become degraded by proteases, most prominently by cathepsins, and where catalyzed
peptide exchange by HLA-DM fulfills its role in the efficient replacement of the invariant
chain-derived peptide CLIP by high-affinity pathogen- or host cell-derived peptides.
Protease action may be limited by protein antigen abundance and redox conditions,
while HLA-DM is regulated at several stages, including by expression levels, pH, or
the co-expression of the competitive inhibitor HLA-DO. HLA-DM activity leads to significant
changes in the immunopeptidome of antigen-presenting cells, thereby tailoring T cell
responses and often shifting antigenicity toward high-affinity immunodominant epitopes
(2). Control of DM activity by DO has been described to be of prime importance in
thymic epithelial cells, in a subset of dendritic cells, and in B cells when entering
the germinal centers for affinity maturation and class switching (3, 4). In all of
these cases, the switch from a broader, self-peptide (CLIP) dominated immunopeptidome
to a more focused repertoire is necessitated by the requirement for more stringent
antigen presentation, often preceding more intense T cell reactivity and proliferation.
Here, we review data on this cellular switch in the functionality of antigen presentation
and propose that it is promoted by an as yet poorly understood molecular switch. Acknowledging
that general biophysical parameters such as pH and redox are important for antigen
processing in general, an elusive DM-DO switch is postulated that would allow rapid
and strong shifts in immunopeptidomes. We capitalize on theoretical considerations
to back our opinion that a regulatable switch would have the advantage of allowing
for a rapid and possibly signal-dependent change in the peptide selection process,
as might be required in the context of rapidly changing immunological conditions.
Regulation of Antigen Processing
Proteolysis of antigens for MHC presentation and T cell surveillance, while certainly
modulated in its efficiency, is thought to constantly report on the proteome state
of cells and organs in the body. It is mostly viruses, bacteria, and cancer cells
that have developed strategies to counter the expression of MHCI or the presentation
of antigens by MHC class I molecules, thereby tuning down the corresponding MHCI immunopeptidomes.
For MHC class II, the situation is more complex. Presentation, in this case, is restricted
to certain types of immune cells, and MHC class II expression itself is regulated
depending, e.g., on the maturation state of a certain cell type (1). Furthermore,
molecules associated with effective MHCII peptide presentation, such as cathepsins,
the exchange catalyst HLA-DM, or its inhibitor, HLA-DO, have been shown to be regulated
in their expression (4). Furthermore, the function of these proteins is pH-dependent,
with an optimum of activity (e.g., for certain cathepsins or the exchange factor HLA-DM,
the pH optimum is close to the acidic pH of the late endosome). We note that HLA-DM
activity differs largely for MHCII allotypes and thus that DM susceptibility can be
truly defined only with regard to a specific peptide-MHCII complex (5). For example,
in the mouse system it has been shown that the I-Ab or I-Ad alleles are strongly dependent
on the mouse homolog of HLA-DM, H-2M, while the E-Ad and E-Ak variants are not (6,
7). However, it seems that even for the latter variants, the exact composition of
the antigen repertoire can be modulated by H-2M (7). Furthermore, for humans it has
been found that several variants of DM exist. Certain combinations of the α- and β-chain
of DM are expressed in the population, and the biochemical and cellular properties
of these DM proteins differ measurably with regard to their activity and pH dependence
and thus result in distinct immunopeptidomes (8, 9).
It has been proposed that redox conditions within the phagolysosomal and endolysosomal
compartments are critical for antigen presentation (10). Here, a balance has to be
maintained between rendering the cysteines of proteases in a reduced, active form
and not reducing the essential disulfide bonds of other proteins as they are present,
for example, in the MHCII molecules themselves. This leaves the conundrum of how certain
antigens can be processed that are stabilized by disulfide bonds but need to be reduced
for efficient digestion by proteases. It appears that enzymes, such as the gamma-interferon-inducible
lysosomal thioreductase (GILT), play an important role (11). For example, it has been
shown that reduction of the house dust mite allergen Derp1 depends on GILT and that
GILT thereby leads to more efficient processing of the protein. Consequently, in a
mouse airway inflammation model of asthma, GILT knockout mice exert mitigated allergic
responses (12). Independent of disulfide reduction, the foldedness of the birch pollen
allergen Betv1 along the endolysosomal pathway has been shown to be critical for its
immunogenicity (13), indicating that protein stability is certainly one of the parameters
that determine the degradation kinetics of a protein and its subsequent loading onto
MHCII molecules. The generally high thermodynamic stability of long-lived MHCII-peptide
complexes might be one reason why they are largely protected from degradation themselves.
However, why the more instable MHCII allotypes such as certain HLA-DQ variants are
shielded from degradation is not clear; presumably membrane partitioning and nanoscale
localization within the late endosome contribute to protecting them.
While the importance of pH, redox conditions, and protein stability for antigen presentation
is undisputed, these parameters describe general biophysical properties that are not
subject to acute control. They rather shape the constitutive process of presenting
peptidomes on MHCII and may tune its general features. Modulation of this constitutive
MHCII pathway by changes in the gene expression of its critical components could impose
long-term control that might be required, for example, during the differentiation
of dendritic cells or B cells. However, the rapid switches of MHCII presentation induced
by antigen- or pathogen-related signals, such as those delivered by Toll-like- or
B-cell receptors (3), are unlikely to rely solely on comparatively slow changes in
gene expression (typically requiring many hours to become manifest at protein level).
Hence, we hypothesize that a molecular switch, operating on shorter “biochemical”
time scales of minutes to hours, is involved in the timely changes of the presented
peptides. First, a cell might signal strong receptor engagement in order to promote
the general turnover of antigen, thereby adjusting its presentation properties to
the new conditions. Thus, sustained exposure to antigen would be distinguished from
more serendipitous events and result in a robust response that precedes irreversible
fate decisions. Molecular switches of this kind would typically operate at the level
of transport or proteolysis. Secondly, a molecular switch could be engaged at the
repertoire level, changing the composition of the immunopeptidome and thereby directly
altering putative T cell responses. This type of switch could comprise site-specific
proteolytic events or modulators of the peptide exchange process itself. In both cases,
altered activity might lead to a shift in the ratio of self-peptides to pathogen-derived
antigen and thus provide a means of rapidly modulating the activation of T cells.
In particular, regulation of the peptide exchange process itself as the most downstream
event in the processing pathway seems to be well-suited to ultimately tuning the presentation
of antigen and stimulation of T cells.
Control of Antigen Exchange by HLA-DM
Antigen loading of MHC class II molecules is a process that depends on the general
features of endolysosomal processing but also capitalizes on molecules uniquely evolved
to enable the highly efficient exchange of placeholder peptides against exogenous,
often pathogen-derived antigens in professional antigen-presenting cells. The placeholders,
termed class II-associated invariant chain peptides, are derived from the invariant
chain (Ii or CD74) (14) as peptides of different length displaying distinct properties
(15, 16) and, for most HLA allotypes, bind via the core sequence MRMATPLLM to MHCII
molecules. CLIP binding to human and mouse MHCII molecules differs widely for individual
allotypes (17), and several mouse alleles (I-Ak, I-Ed, I-Ek) (6, 7), as well as human
allotypes (e.g., HLA-DQα1*0501/DQβ1*0301) (18, 19), show poor CLIP binding, and thus
peptide replacement takes place efficiently even in the absence of H-2M/HLA-DM. However,
the exchange catalyst for these alleles still seems to be required to stabilize unoccupied
MHCII molecules or efficiently shape the repertoire toward higher affinity peptides
(7). Interestingly, the CLIP sequence can bind in two flipped orientations along the
MHCII binding groove, with the equilibrium of the canonical vs. inverted binding mode
depending on the length of the N- and C-terminal overhang regions, a process that
itself is catalyzed by HLA-DM (20, 21). In any event, the CLIP peptides of HLA-DM/H-2M
susceptible allotypes are replaced in the late endosomal compartments by other peptides
with similar or higher affinity. Alternatively, if large concentrations of a non-optimal
antigen are provided, it might also be loaded onto the MHCII molecule, simply based
on the law of mass action. However, since many MHCII-CLIP complexes, especially those
of the HLA-DR locus, are already of high kinetic stability, replacement rates are
very low for these HLA allotypes and would not proceed to a significant degree on
a physiologically relevant timescale (minutes-to-hours). The exchange catalyst HLA-DM,
which has its activity optimum at or near the acidic pH of the late endosome, leads
to more efficient exchange even for MHCII allotypes that have low CLIP off-rates (2).
Peptide exchange occurs via rare conformation in the HLA molecules, which occupy at
most a few percent of the conformational ensemble and are recognized by HLA-DM (22,
23). This mechanism ensures that, for stable pMHCII complexes, the peptide can still
be replaced by higher affinity ligands within a time frame of seconds to minutes.
A simple mathematical model based on experimentally determined exchange rates (22)
shows how the HLA-DM to HLA ratio controls the switching time (Figure 1). A somewhat
sub-stoichiometric ratio of DM:MHCII in the range of 0.1–0.3 still enables switching
times within the order of minutes (Figure 1). Thus, the active concentration of the
peptide exchange catalyst HLA-DM controls switching time.
Figure 1
HLA-DM concentration controls the rate of peptide exchange at HLA. We modeled how
rapid transitions in the conformational ensemble of HLA-peptide are modulated by HLA-DM
binding; HLA-DM stabilizes a rare active conformational state that facilitates peptide
exchange [see Figure 1E in Wieczorek et al. (22)]. Based on the experimental data
obtained from Wieczorek et al. (22), the modeling shows that HLA-DM concentration
controls the switching time most strongly in the substoichiometric regime.
Indeed, the ratios of DM to MHCII in primary cells have been reported to be quite
variable and to depend on the differentiation state of professional antigen-presenting
cells (16). However, the curve in Figure 1 also argues that the system does not need
to operate at a stoichiometric steady-state level for efficient catalysis, leaving
open the question to what extent expression levels have to change in order to modulate
the presented repertoire significantly. Experimentally, it has been shown that a significant
minority of peptides and epitopes change when DM levels are raised from low to high
in a cellular model (2). In particular, the amount of high affine peptides is fostered
in the presence of HLA-DM, a consequence that might be favorable in a certain immunological
context but undesired in others. Therefore, regulation of DM activity is a key issue
when considering global shifts in immunopeptidomes during MHCII-mediated T cell responses.
Discussion–Switching off Catalyzed Peptide Exchange
Biasing the repertoire of MHCII bound peptides toward high-affinity ligands might
be harmful or advantageous, depending on the immunological context [reviewed in Alvaro-Benito
et al. (24)]. For example, it has been found that the absence of HLA-DM in mice in
the context of a type I diabetes model prevents the animals from acquiring the disease
(25), while, on the other hand, DM seems to be required for constraining bacterial
pathogens such as Mycobacterium tuberculosis (26). Moreover, DM expression in the
thymus has been found to be low in the cortical but high in the medullary epithelial
cells of the thymus (27), indicating that positive and negative selection have distinct
requirements for DM during T cell development. Apart from the modulation of DM activity
during these processes by regulation, DM gene expression downregulation of HLA-DM
can also be achieved by co-expression of the DM competitive inhibitor HLA-DO (DO).
DO binds with much higher affinity to DM than canonical MHCII molecules, thereby fully
abrogating DM exchange activity when present at stoichiometric concentrations (28,
29). DO has also been suggested to have a direct effect on classical MHC class II
molecules by recognizing a receptive conformation of the common allotype HLA-DR1 (30),
and it will be interesting to see whether the underlying rare conformations can be
detected directly by experiment. More recently, a comprehensive immunopeptidome study
was performed comparing DO knockout and wild-type human lymphoblastoid HLA-DR1 homozygous
LG-2 cell lines, extending previous studies on DM-independent peptide loading (6,
7) and corroborating the finding that DO broadens the repertoire, thereby counteracting
the effect of DM to a certain extent (4). How far such broadening of the repertoire
plays a role during certain phases of murine B cell development and dendritic cell
differentiation, two processes where DO expression is known to be high, is an intriguing
question. In human B cells, DO levels are high only when B cell development in the
bone marrow is complete and are then downregulated in germinal centers, where affinity
maturation of B cells proceeds and where T/B cell cooperation becomes of critical
importance (31, 32). March9-mediated ubiquitination is mostly thought to be responsible
for the reduced DO levels in GC B cells (33), but this leaves open the question of
how March9 activity is itself regulated. The DM-DO complex is extremely stable and
pH insensitive when investigated in vitro (KD = 3.7 nM). However, once dissociated
from DM, DO is rapidly inactivated at acidic pH, and an indirect influence of acidification
on DO stability along the endolysosomal pathway has therefore been suggested (34).
The degradation rate of DO can be modeled based on the KD of the complex. Assuming
a typical kon for protein-protein interactions of 105 M−1 s−1 (35), the calculated
half-time for complex dissociation is ~30 min. Assuming that each dissociation event
will translate into conformational changes and subsequent degradation of the protein,
this time represents the lower limit for DO downregulation. In cells, there will be
some degree of competition between fast rebinding of acutely dissociated DO to DM
and DO degradation, so that the effective half-life for DO is likely to be longer.
Indeed, in vitro studies showed that concentrations of free DM and its associated
exchange activity after preincubation of DM:DO complexes at acidic pH were significant
only after 2 h and free DM was still increasing after 24 h when analyzing loading
of the hemagglutinin peptide HA in cellular lysates (34). Thus, it seems that acidification
alone would lead to a slow, gradual, and non-reversible increase in DM activity.
Rather than degradation of DO after slow release from a tight complex with DM, the
lowering of the effective DO-DM affinity would provide a much faster means to release
DM activity from DO inhibition and, in turn, switch the HLA-presented peptidome. Moreover,
this switch would be reversible as long as DO is present, enabling the system to adapt
to changing environmental stimuli. What is the evidence that such a switch would be
advantageous in vivo? There is no direct evidence yet that the DM-DO complex is reversibly
and rapidly switchable. However, circumstantial findings for adaptive changes in antigen
presentation exist where fast control of the DM:DO ratio would be the most straightforward
manipulatable parameter to change the peptide repertoire. As mentioned before, DO
expression is seen in B cells first when they transition from the immature to the
mature state. These cells then enter the germinal centers, and as earlier studies
by Jensen and coworkers (32) showed, stably express DO in the initial IgD+CD38− B
cell population that has not yet undergone affinity maturation. Interestingly, GC
B cells of the IgD−CD38+ type, which should consist of centrocytes and centroblasts,
downregulate DO (32). Since these cells, especially the centrocytes in the light zone,
are in intense contact with follicular TFH cells, DO downregulation is anticipated
to unleash DM activity in order to allow the display of high-affinity pMHCII complexes
that are in turn prone to engage more robustly in sustained T cell activation and
thus provide B cell help. Interestingly, a third GC B cell population, namely IgD−CD38−,
thought to represent memory cells, shows robust DO expression levels. Thus, it is
clear that interconverting B cell populations exist that are under selective pressure
to encounter high-affinity antigen (36). While changing DO levels reflect this requirement,
it is likely that the genetic control is supported by regulation at the protein level.
Ubiquitination-dependent degradation surely represents a possible regulatory mechanism
(33), but it has the disadvantage of being irreversible and energetically costly.
A reversible switch would, for example, allow the individual GC B-cell populations
to interconvert prior to an irreversible fate decision. Moreover, when coupled to
B cell receptor activation, a more robust “two-signal” scenario could be envisaged,
where B cell receptor signaling by a higher affine B cell receptor is intertwined
with more persistent presentation of a high-affinity pMHCII complex. There is experimental
evidence that DM interacts with endocytosed B cell receptor in the late endosome (37),
and it is thus not unlikely that such a coupling exists. It will be important to test
whether the DM/Ig binding is altered in the presence of DO, since this could indicate
that the BCR could act as a switch itself or at least contribute to a more acute response
during B cell antigen presentation.
What could be the nature of this switch? Is it possible that a proteolytic event is
coupled to such a switch in activity? It is well-known, for example, that invariant
chain processing is a processive event where cleavage proceeds from the C-terminal
to the N-terminal end (38–40). In particular, Cathepsin-S is critical for the production
of the N-terminal fragments (41, 42) that could in principle be involved in the appearance
of peptides other than CLIP and that may exert unanticipated functions through binding
to canonical or non-canonical MHCII molecules. The fact that at least a partial MHCII
binding groove exists in DO makes it at least conceivable that a proteolytically cleaved
peptide binds to it (28).
This peptide would have been identified if it was constitutively present or if several
peptides could bind via anchor residues as is the case for canonical MHCII molecules.
Rather, this peptide would be produced under certain conditions in a switch-like manner
that also allows its fast removal once DM is bound again or when ubiquitination-mediated
degradation ensues. The proteolytic hypothesis, stating that inducible cleavage of
a protein fragment results in a competitive DO binder, could be tested by applying
selective inhibitors against, for example, cathepsin S (43) and analyzing the amount
of free vs. bound HLA-DM. Alternatively, the spectrum of post-translational modifications
of free vs. DO-bound DM molecules could be revisited, as there is ubiquitination,
phosphorylation, glycosylation, and lipid modification. Notably, the DM α-chain contains
a putative palmitoylation site in its short cytoplasmic tail (44) that could serve
as a signal in changing the nanoscale localization of the molecule. Similarly, glycosylation
and phosphorylation sites have been identified in the DO β-chain (45, 46). Capitalizing
on modern, highly sensitive mass spectrometers, previously unobserved changes might
be captured that are physiologically relevant and that could be validated by corresponding
site-specific mutations. Together with the option that Ig binding could also act in
a manner dependent on DM-DO complex-formation (37) (see above), we thus present three
experimentally testable conditions to reinforce or disprove the suggestions made in
this opinion article.
Assuming the existence of such a molecular event, it will quickly release inhibition
of DM by DO and thus will lead to a temporally regulatable activity of the exchange
catalyst. As the concentration of active DM not only controls the speed of peptide
exchange but also affects the affinity range of presented peptides in an as yet poorly
understood manner (see above), such a molecular switch would be an important regulator
of the quality and quantity of antigen presentation by MHCII. It is worth pointing
out that antigen recognition, at the T cell end of the interaction with the APC, also
employs an active molecular switch: T cells do not simply sense the affinity of the
interaction of peptide-loaded MHC with the T cell receptor but rather employ kinetic
proofreading to discriminate between their agonists and self-peptides (47, 48). Thus,
in a conceptual framework, the regulation of antigen presentation on the APC may not
be dissimilar from antigen recognition by T cells in employing active molecular switches.
Author Contributions
CF and TH drafted and wrote the manuscript together.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.