Joint Bone Spine 88 (2021) 105164

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Review

Antigen-specific tolerance approach for rheumatoid arthritis: Past,

present and future
Audrey Page , Floriane Fusil , François-Loïc Cosset ∗
CIRI – Centre international de recherche en infectiologie, Université de Lyon, Université Claude-Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308, ENS Lyon,
46, allée d’Italie, 69007 Lyon, France

a r t i c l e i n f o a b s t r a c t

Article history: Rheumatoid arthritis is a chronic systemic autoimmune disease, affecting mainly the joints. It is caused
Accepted 2 February 2021 by an adaptive immune reaction against self-antigens, leading to the over production of inflammatory
Available online 19 February 2021 cytokines and autoantibodies, mainly mediated by autoreactive CD4+ T cells and pathological B cell
clones. The treatment options currently available rely on palliative global immunosuppression and do
Keywords: not restore tolerance to self-components. Here, we review antigen-specific tolerance approaches that
Rheumatoid arthritis have been developed to inhibit or delete autoreactive clones, while maintaining a potent immune sys-
Antigen-specific tolerance
tem for rheumatoid arthritis. The first attempts relied on the oral ingestion of self-reactive peptides,
Self-peptides
Tolerogenic cells
with lukewarm results in human clinical trials. To enhance treatment efficacy, self-peptides have been
Anergy engineered and combined with immunosuppressive molecules. In addition, several routes of delivery
have been tested, in particular, nanoparticles carrying self-antigens and immunomodulatory molecules.
More recently, transfer of immune cells, such as tolerogenic dendritic cells or regulatory T cells, has been
considered to restore tolerance. Although promising results have been obtained in mouse models, the
translation to humans remains highly challenging, mainly because the disease is already well developed
when treatments start and because patient’s specific self-antigens are often unknown. Nevertheless,
these approaches hold great promises for long-term RA treatment.
© 2021 Société française de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.

1. Introduction This inflammation is caused by a rupture of tolerance. Even if the

Rheumatoid arthritis (RA) is an autoimmune disease that affects
ca. 0.5% to 1% of the population worldwide. Typical patient symp-
toms range from morning stiffness, pain, and difficulties to walk
to life-threatening complications, such as cardiac or pulmonary
issues. The disease is characterised by an immune reaction against
self-antigens that leads to chronic systemic inflammation. The
synovial tissue of joints is particularly inflamed, which trigg-
ers subsequent erosion of cartilage and bone driven by matrix
metalloproteinases, produced locally by macrophages and syno-
vial fibroblasts following pro-inflammatory cytokines exposure
[1]. Interestingly, anti-inflammatory cytokines, such as IL-10 and
TGF-␤, can counteract pro-inflammatory action, by inhibiting
MMP production and also by inducing native inhibitors of MMPs
[2].
∗ Corresponding author.
E-mail address: flcosset@ens-lyon.fr (F.-L. Cosset).
initial trigger of RA is not clear and surely multifactorial, genetic,
environmental factors, smoking and buccal infections by several
pathogens have been associated to breakdown of tolerance [3,4].
RA patients have defective central and peripheral tolerance check-
points, which may promote the development of auto-reactive B and
T-cell clones that escape the negative selection mechanisms and are
detected in RA patients [5]. These autoreactive B cells actively pro-
duce pathological autoantibodies that can be used as disease and
prognosis biomarkers [6]. For RA, autoantibodies are divided into
two main categories: rheumatoid factors (RFs), which recognise
the Fc portion of immunoglobulins, and anti-citrullinated protein
antibodies (ACPAs) [7]. ACPAs recognise post-translationally modi-
fied self-proteins, including vimentin, filaggrin, collagen, fibrinogen
and tenascin-C, in which arginine residues have been converted
to citrulline [8,9]. Although citrullination of proteins is a nor-
mal process that is catalysed by the peptidyl-arginase deiminase
(PAD) enzyme, the development of autoimmunity against these
citrullinated antigens marked by ACPAs is not normal [10]. Of
note, activated PADs have been shown to be released in neu-
trophil extracellular traps (NETs) [10]. NETs are DNA filaments

https://doi.org/10.1016/j.jbspin.2021.105164
1297-319X/© 2021 Société française de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.
A. Page, F. Fusil and F.-L. Cosset
entangled in proteins that are released in the extracellular
space [10]. They are produced by polymorphonuclear neutrophils
and are believed to display pro-inflammatory molecules and
auto-antigens (that can be citrullinated in situ), which would
enhance epitope spreading and autoimmunity, thus worsening the
disease [10].
Among auto-antigens associated to RA, one of the most stud-
ied is type II collagen (Collagen II). Anti-collagen II autoantibodies
directed either against native triple helical or citrullinated epi-
topes are present in the serum, synovial fluid and cartilage of
respectively 10% and 40% of RA patients [11]. The immunisation
of mice with collagen II leads to pathological manifestations that
mimic several aspects of RA and represents a model called collagen-
induced-arthritis (CIA) that is frequently used. Although the exact
pathogenic role of citrullinated collagen II in human RA patients
is not known, this CIA model has been widely used to study RA
pathogenesis and to develop new treatments.
Current treatments for RA rely on global immunosuppressant
drugs to control inflammation by targeting downstream effectors
of autoimmune responses. Non-steroidal anti-inflammatory drugs
(NSAIDs), glucocorticoids or disease-modifying anti-rheumatic
drugs (DMARDs), such as methotrexate, are usually indicated as
first-line medications [12]. New treatments have driven significant
improvements, such as anti-inflammatory drugs, anti-cytokine
therapies, monoclonal antibodies (anti-TNF␣), and T-cell or B cell
inhibitors, and, recently, inhibitors of cytokine signalling (JAK
inhibitors) [12]. Although these therapeutic molecules alleviate
patient’s symptoms, and consequently improve their quality of life,
they do not address the causes of the disease. Consequently, nei-
ther long-term drug-free remission nor restoration of tolerance to
self-antigens can be achieved, and lifelong treatments are required.
Therefore, there is an obvious need for new RA treatments that
could specifically target the causes of the disease, thus reducing
side effects. In this context, re-inducing self-antigen-specific toler-
ance has emerged as an attractive strategy to control both cellular
and humoral autoimmune responses.
Importantly, an ideal immunotherapy cure would reprogram
the immune system to a state of self-tolerance and lead to disease
remission by specifically impacting pathological immune reactions
and cells. Indeed, a potent protective immunity against pathogens
and cancer cells, for instance, should be permanently maintained
during and after the treatment. Such approaches aim mainly at
modulating T cell subpopulations, either by depleting autoreactive
CD4+ Th1 and Th17 cells or by inhibiting their effector functions
through the expansion of self-antigen-specific regulatory T cells
(Tregs). The ratio of Treg and Th17 cells has emerged as a new
paradigm for RA [13]. Indeed, although Treg counts are heteroge-
nous in RA patients, their function is impaired and, hence, they fail
to inhibit the activation of pathologic immune cells, notably T cells
[13]. In addition, autoreactive B cells have key pathological func-
tions, not only as antigen-presenting cells, but also by mediating
direct inhibition of autoreactive T cells or conversion to regulatory
B cells, hence resulting in decreased amounts of autoantibodies.
This would also reverse the ratio of pro-inflammatory cytokines,
such as IL-1 and TNF-␣ over anti-inflammatory cytokines, such as
IL-10 and TGF-␤, and ameliorate disease manifestations. Indeed,
these two latter cytokines can induce Tregs and promote tissue
repair.
However, some limitations are intrinsically related to such tech-
nologies. Indeed, even if some self-antigens commonly associated
to RA are known, the diseases causing self-antigens are often dif-
ferent among patients and evolve by epitope spreading during the
course of the disease, so the treatments need to be adapted. Nev-
ertheless, antigen-specific tolerance approaches hold promise for
longer-term efficacy, greater specificity and lower toxicity for RA
treatment.
2
Joint Bone Spine 88 (2021) 105164
Here, we review the main approaches that have been under-
taken over the past years to restore antigen-specific tolerance in
RA.
2. Native self-peptides administration
Oral tolerisation is probably the most studied approach for
restoration of antigen-specific tolerance since the first attempts
date from the beginning of the 1990s (Fig. 1). This approach
takes advantage of the natural tolerisation processes occurring
when food is ingested, which prevents allergy in physiological
context. Several barriers restrict macromolecule ingestion across
the intestinal epithelium, such as proteases and mucus. M cells,
which are located in the Peyer’s patches of the gut-associated lym-
phoid tissue (GALT), are specialised in the transport of antigens
by transcytosis across the mucosal barrier. After transcytosis, anti-
gens are loaded on antigen-presenting cells, such as dendritic cells
(DCs), and may interact with other immune cells, such as T cells.
These cells then migrate to mesenteric lymph nodes and reach the
systemic immune circulation before being recruited at inflamed
areas. Depending on the dose of oral self-peptide intake, different
tolerance mechanisms can be induced: anergy of autoreactive T
cells, deletion of peripheral antigen-specific T cells, or induction
of Tregs, for high or low doses of antigens respectively [14,15].
Indeed, stimulation with high doses of self-reactive peptides blocks
T-cell proliferation and IL-2 production by immune cells or cause
activation-induced cell death. In contrast, low peptide doses may
lead to antigen-driven suppression of autoreactive clones by induc-
tion of Tregs in the GALT, which then migrate to the systemic
immune system and to inflamed areas, such as the osteoarticular
joints [14–16]. These cells mediate their immunosuppressive func-
tions through secretion of anti-inflammatory cytokine, such as IL-4,
IL-10 and TGF-␤. Interestingly for autoimmune disease therapy,
low-dose self-antigen intake may also suppress immune responses
unrelated to the ingested self-peptide by bystander effects, since
Tregs primed in an Ag-specific manner can drive Ag-unspecific
suppressions [16].
Several self-antigens, such as bovine or chicken collagen II,
HCgp39 and dnaJp1, have been investigated for restoration of oral
tolerance in RA. Ingestion of collagen II peptides or whole colla-
gen II proteins markedly reduced arthritis incidence and severity in
collagen-induced arthritis (CIA) mice [14,17,18]. While most stud-
ies have focused on prophylactic effects of oral treatment, i.e. by
starting it before or concomitantly to animal immunisation, one
study showed a beneficial impact on disease severity in a ther-
apeutic setting, paving the way for human diseases translation
[18]. However, results from human clinical trials after self-peptide
ingestion have been quite disappointing so far and only led to
minor improvements of rheumatic scores as compared to control
or placebo treatment [19–24].
Further clinical trials are required to assess the efficacy of these
approaches. Nevertheless, oral treatments with self-antigen pep-
tides are globally well tolerated by patients with minor adverse
events, and their efficacy might be enhanced by determining the
optimal doses, frequency of injection, formulation of self-peptides,
and by combination with current anti-rheumatic drugs.
Even if oral ingestion is a very convenient way for
self-peptide delivery, several other routes of administra-
tion have been tested, such as direct gastric application of
self-peptides [25,26] or nasal application [27,28]. The more com-
monly used route to deliver most vaccines is via intradermal
injections, which also showed particularly interesting results
for CIA [29]. The intravenous route has also been considered for
self-peptides infusion and induced a state of self-tolerance after
immunisation [30]. One potential explanation for this lack of
A. Page, F. Fusil and F.-L. Cosset Joint Bone Spine 88 (2021) 105164

Fig. 1. Mechanisms of oral tolerance in RA. Self-peptides or proteins are orally given to the patient. In the GALT (gut-associated lymphoid tissue), and more particularly in
Peyer’s patches, self-peptides are transcytosed by M cells, and loaded on antigen presenting cells (APCs), such as dendritic cells. Autoreactive T cells interact with antigen
loaded APCs and may either undergo anergy or deletion or will differentiate into regulatory T cells. These Tregs migrate to mesenteric lymph nodes, where they can reach
other immune cells and inhibit the autoreactive clones. Autoreactive regulatory cells (B and T cells) then regulate the autoimmune inflammation at the joints by inhibiting
autoantibody secretion and restoring the balance between Th1/17 and Treg/Th2 that respectively secrete pro and anti-inflammatory cytokines.

clinical efficacy might be the short half-life of self-peptides and,
consequently, several additional injections might be required to
reach a therapeutic effect.
3. Gene therapy
One of the main hurdles with the above-mentioned approaches
is that repeated injections of high doses of tolerogen molecules,
such as native self-peptides, might be required. This represents
elevated costs and inconvenience to patients, due to recurrent pro-
cedures. To circumvent these issues, several strategies have been
developed to induce in vivo expression of tolerogenic self-peptides.
Firstly, DNA vaccines encoding collagen II yielded signifi-
cant improvements in CIA mouse models after intramuscular or
intravenous injection [31,32]. Joint destruction, pro-inflammatory
cytokines and epitope spreading have been significantly decreased
following such vaccination [31].
Alternatively, viral vectored gene therapy with lentivirus, ade-
novirus or vaccina virus has been applied to express in vivo
autoantigen and cure RA [33–37]. For instance, the intravenous
infusion of lentiviral vectors, encoding the immuno-dominant epi-
tope of CII in the CLIP peptide, not only prevented the development
of the pathology in a prophylactic setting, but also greatly reduced
disease severity in a therapeutic approach [35–37]. These bene-
ficial effects were associated to a decrease in collagen II-specific
antibodies and an increase in the percentage of Tregs [35,36].
Although the induction of self-peptides in vivo to restore toler-
ance seems a promising approach, to date, this strategy cannot be
widely developed for RA, especially because the post-translational
modifications of self-peptides, such as citrullination, cannot be
encoded genetically. Consequently, these peptides need to be
exogenously produced with the correct modifications and then
infused. Before infusion, these self-peptides can also be engineered
to enhance their tolerogenic properties or to promote their delivery
to the right cells and at the right place.
4. Infusion engineered bio-compounds
4.1. Self-peptide fusion molecules
Several concepts have been tested to enhance the tolerogenic
potential of infused self-peptides, either by modifying their epi-
topes to render them more immunosuppressive, by combining
several self-peptides together or by fusing them with immunosup-
pressive moieties.

3
A. Page, F. Fusil and F.-L. Cosset Joint Bone Spine 88 (2021) 105164

Some native regions of self-proteins have been shown to
exhibit immunosuppressive properties, such as a portion of bovine
collagen II that induces an immune response against an immuno-
dominant region of the TCR [29]. However, self-peptides can also
be engineered in order to suppress pathological immune responses
while maintaining antigen specificity. For instance, one study
reported a recombinant collagen II peptide harbouring three point
mutations (in residues critical for antigen presentation and thus
T-cell activation), which drastically suppressed immunity to colla-
gen II after co-immunisation with native collagen II [38] or after
intra-articular tolerogen peptide gene transfer [34].
As an alternative approach, multi-epitope fusion peptides have
been created, which expands the array of epitopes against which
tolerance is sought and counteracts epitope spreading, a process
that widens the range of closely related self or not-self peptides
recognised by the immune system. Such multi-epitope molecules
can be composed of several epitopes from the same protein, such as,
e.g. a recombinant peptide containing two tolerogenic epitopes of
chicken collagen II [39], or of epitopes from different self-proteins
[40]. For instance, a synthetic fusion protein incorporating cit-
rullinated epitopes of vimentin, fillagrin, fibrinogen and collagen
II reduced arthritis severity to a much greater extent than the
mono-epitope peptide [40]. This was mediated by decreased infil-
tration of immune cells in the joints and by generation of Tregs
[40].
Finally, immuno-modulators, such as tolerogen molecules or
activation inhibitors, e.g. immune checkpoints inhibitors, have
appeared as good candidates for linkage to self-antigens. Fusion
molecules have been created to exploit the tolerogenic potential of
antigen presentation either by direct targeting (with anti-DEC205
antibodies to induce its delivery to DCs) [41] or by compelling pre-
sentation on MCH II (with linkage to human ␤2 microglobulin)
[42].
4.2. Nanoparticles
Although molecular engineering has greatly improved the
tolerogenic potential of self-peptide drugs, a major limitation
impairing their clinical success, is the route of delivery. Indeed,
following injection, such molecules are rapidly degraded. Nanopar-
ticles appear as promising platforms for drug delivery and have
been tested for self-peptide carriage (and protection from inhi-
bition by circulating autoantibodies), to restore tolerance in RA.
Several approaches based on nanoparticles have been explored for
re-induction of tolerance (Fig. 2).
Firstly, antigen-presenting cells can be targeted by nanopar-
ticles as nanoparticles are rapidly cleared by innate immune
phagocytes. For instance, the oral ingestion of polymer contain-
ing collagen II has greatly reduced the incidence and the severity
of CIA, as well as the autoantibody titers, compared to control
groups [43]. Treatment efficacy may be further enhanced by com-
bining immune-modulators on the nanoparticle, such as cytokines,
toll-like-receptor (TLR) ligands or rapamycin, which can promote
cell differentiation toward more tolerogenic phenotypes. Remark-
ably, the co-delivery of NF-␬b inhibitors and self-peptides loaded
on liposomes reduced clinical signs of the disease in mice [44].
Similarly, calcitriol addition in liposomes has shown encouraging
results in a proteoglycan (PG)-induced mouse model, as it sup-
pressed the expansion and function of Teffs, and induced Tregs
[45].
Secondly, autoreactive B and T lymphocytes can be directly
targeted and inhibited. For inhibition of autoreactive B cell, self-
peptides can be directly bound on the nanoparticle surface along
with cell-targeting and immunosuppressive ligands. For example,
the coupling of citrullinated self-peptides with CD22, an immune
inhibitor [46], or with complement-activating lytic peptide [47]
was shown to selectively inhibit pathological B cells. For inhibition
of autoreactive T cell, nanoparticles can harbour MHC complexes on
the nanoparticle surface that present self-peptides in the absence
of co-stimulation, which may induce suppression of effector
function.
Overall, nanoparticles represent an auspicious method to inhibit
autoimmune responses either by acting directly on pathologic lym-
phocytes or by presentation on APCs; yet, clinical trials are required
to test their safety and efficacy at a large scale before this therapeu-
tic approach become widely available.

Fig. 2. Antigen-specific tolerance restoration by nanoparticles. Two main approaches for restoring tolerance can be used with nanoparticles. Firstly, APCs can be targeted.
In this case, nanoparticles contain or harbour self-peptides (which will be phagocytosed and presented on MHC-II molecules), sometimes in combination with immuno-
modulators (which will inhibit pathologic inflammation), restoring tolerance to self-peptides. Secondly, autoreactive adaptive lymphocytes can be targeted. For autoreactive
T cell inhibition, nanoparticles can harbour MCH-self-antigen complex with checkpoints co-inhibitors, while for autoreactive B cell inhibition, self-peptides can be directly
bound to nanoparticle surface. Although nanoparticles are naturally endocytosed by phagocytes, antibodies or receptors recognising cell surface molecules can be added on
the nanoparticles to target specific cell types.

4
A. Page, F. Fusil and F.-L. Cosset
5. Infusion of engineered immune cell
5.1. Engineering dendritic cells
Besides nanoparticles, cells may also directly be used to carry
self-antigens, after ex vivo loading and re-infusion. DCs repre-
sent favourite targets for such purposes as they are professional
antigen-presenting cells that naturally migrate from peripheral
tissue to lymph nodes, where they interact with T cells through
the presentation of antigen in a human leukocyte antigen (HLA)
– restricted manner. This interaction can either activate or inhibit
immune responses against the presented antigen, depending on
the DC phenotype. Immature and mature endogenous DCs can-
not be directly re-injected after self-peptide loading, since they
would actually cause immunogenicity instead of tolerance. Sev-
eral approaches have been explored to skew maturation of DCs
toward induction of a tolerogenic state (TolDCs) ex vivo before
their reinfusion by specific culture conditions [48]. These tolero-
genic effector functions ameliorate RA disease manifestations
after adoptive transfer in mouse models through reduction of
Th17 cells, suppression of autoreactive T cells, specific induc-
tion of Tregs and switch in the Th1/Th2 balance in favour of Th2
[49,50].
Although promising results have been obtained in several
mouse models, their translation to humans is highly challenging,
mainly because of the costs and the wide range of available methods
to modulate DC functions and produce clinical grades of either allo-
genic or autologous tolDCs. Three main phase I clinical trials have
been launched to test the efficacy of TolDC therapy in RA patients
(Table 1).
One study used autologous tolDCs differentiated ex vivo from
peripheral blood monocytes of RA patients, before loading with
four citrullinated self-peptides. RA patients then received a sin-
gle intradermal injection of this compound (called “rheumavax”)
[51]. Rheumavax was well tolerated and appeared to be safe [51].
In addition, after one month of treatment, the ratio of Tregs to Teff
cells was significantly improved and the serum levels of certain
pathological cytokines and chemokines were decreased in patients
[51].
Semi-mature autologous DCs pulsed with recombinant protein-
arginine deiminase type-4, heterogeneous nuclear ribonucleo-
protein A2/B1, citrullinated filaggrin and citrullinated vimentin
antigen have been alternately tested in RA patients [52]. The
patients who received this cocktail (designated as CreaVax-RA)
exhibited decreased numbers of interferon (IFN)-␥-producing T
cells and autoantibody titers [52]. Of note, no clinical efficacy was
observed for patients without any autoantibodies [52].
Table 1
Phase I clinical trials with TolDC to cure RA.
Trial Rheumavax
Number of patients 29
DCs Autologous modified with a
nuclear factor kB (NF-kB)
inhibitor
Antigen(s) Citrullinated peptides
Number of infused cells Low dose: 7.2 × 103 to 1.7 × 104
High dose: 2.7 × 104 to
6.2 × 104
Number of injection(s) 1 5
Route of infusion Intradermal
Monitoring 4 weeks to 6 months
Clinical outcomes Improvement of the ratio
Treg/Teff cells
Reference [51]
5
Joint Bone Spine 88 (2021) 105164
In the two above-mentioned clinical trials, the intradermal
and the subcutaneous routes were respectively tested, while the
AuToDeCRA trial assessed the safety of another route for tolDC
injection, namely by intra-articular administration [53]. TolDCs
were differentiated from patient’s monocytes and pulsed with
synovial fluids containing self-antigens, before injection in the
inflamed knee [53]. Although a decrease of arthroscopic symp-
toms was observed in some patients, no systemic effects could be
detected [53].
Overall, several types of self-antigens, doses of infused cells
and routes of injection have been tested in phase I clinical tri-
als, demonstrating the safety of such approaches but only driving
minor or no improvements in rheumatic patients (Table 1). These
limited improvements of the clinical scores might result from
the status of these autoimmune arthritis patients, in whom dys-
regulated immune responses are already established, sometimes
for years. Further studies are required to really assess the clin-
ical efficacy and antigen-specific effects of TolDC therapy in RA
patients. Interestingly, combinations with anti-rheumatic drugs
may act synergistically and potentiate treatments with TolDCs
[54].
5.2. Engineering lymphocytes
As previously mentioned, pathological lymphocytes recognising
self-components are present in RA patients. Autoreactive B cells are
indeed major drivers of the pathology since B-cell deficient mice do
not develop CIA following immunisation [55]. Furthermore, B cell
depletion by monoclonal antibodies has demonstrated strong pos-
itive impacts on disease courses in RA patients [56]. Conversely,
B cells can exhibit tolerogenic properties, which may ameliorate
disease manifestations. For instance, one study also attempted to
take advantage of the natural antigen-presenting capacity of B cells.
By transducing hematopoietic stem cells before engraftment with
a lentiviral vector encoding a collagen II inside the CLIP peptide,
presentation of collagen II epitopes on MHC-II complexes was com-
pelled. This presentation produced a tolerogenic effect and reduced
the incidence and severity of the pathology [57].
Alternatively, Tregs, as mentioned above, represent a subset of
T cells that exhibit immunosuppressive functions by suppressing
inflammation in an antigen-specific manner without impacting the
protective immune responses. Furthermore, Tregs have the ability
to home towards inflammatory sites, recognise their specific anti-
gens presented on APCs and locally suppress inflammation through
the secretion of cytokines or the expression of immunosuppress-
ive surface molecules. Consequently, adoptive transfer of Tregs
has been considered to restore tolerance and hence, to achieve
CreaVax RA AuToDeCRA
12 13
Autologous semi mature Autologous TolDCs
Recombinant PAD4, RA33, Synovial fluid antigens
citrullinated-filaggrin and
vimentin antigens
Low dose: 0.5 × 107 Low dose: 1 × 106
High dose: 1.5 × 107 Medium dose: 3 × 106
High dose: 10 × 106
1
Subcutaneous Intra-articular
14 and 24 weeks 1, 2 and 13 weeks
Decrease of IFN-g producing Decrease of arthroscopic
cells synovitis symptoms
Decrease of auto antibodies No systemic effects
[52] [53]
A. Page, F. Fusil and F.-L. Cosset
remission in several autoimmune diseases, including RA. Although
adoptive transfer of non-specific or self peptide-specific Tregs has
improved clinical symptoms in CIA, autoantigen specific Treg cell
transfer (Hsp60–specific CD8+ or collagen-II-specific type 1 Tregs)
also mediated reduction of disease [58,59].
The clinical efficacy of Tregs transfer has been hampered by the
high amount of pro-inflammatory cytokines in the joints, which
could not be counterbalanced by the infused cells. Another lim-
itation of Tregs lies in the difficulty to characterise and expand
these cells. Although some methods were developed to expand
autoreactive Tregs, such as their exposure to autoantigen-loaded
APCs, producing high amount of antigen-specific Tregs remains a
major challenge [60]. To circumvent the small number of autore-
active Tregs that can be retrieved and expanded, Tregs may be
genetically engineered in order to redirect their specificity against
self-antigens. Accordingly, ectopic autoreactive T-cell receptor
(TCR) or chimeric-antigen receptors (CAR) can be expressed after
gene transfer in Tregs. Using this latter method, adequate numbers
of auto-antigen specific TCR or CAR-engineered Tregs can easily
be manufactured. Paving the way for translation in autoimmune
disorders, Fransson et al. expressed a CAR targeting myelin oligo-
dendrocyte glycoprotein along with FoxP3 in T cells, which, after
intranasal application, could suppress an ongoing encephalomyeli-
tis [60].
Alternatively, T cells may also be engineered to directly suppress
only pathologic B cells. Toward this goal, novel chimeric receptors,
referred as BAR – for B-cell antibody receptor, have been gener-
ated by replacing the scFv of the CAR by the self-antigen itself [61].
Promising results with transfer of BAR-engineered T cells leading
to inhibition of antigen-specific B cells in the context of hemophilia
[62] indicate that BARs may also be used for RA.
Overall, with the advances in proteomics, gene engineering
and editing techniques, cell therapy with engineered lymphocytes
represents a very appealing approach to cure RA. Nevertheless,
important questions remain to be considered, such as the phys-
iological consequences of modified cell infusion, as well as the
long-term persistence of edited cells, their re-activation potential
and their differentiation.
6. Conclusion and perspectives
Because of their potentially life-long therapeutic effect, sev-
eral strategies to restore antigen-specific tolerance have emerged
towards remission of RA. The first attempts relied on the oral inges-
tion of self-peptides, with promising results in animal models.
However, clinical trials failed to meet the expected improvement
criteria of clinical scores. Self-peptides have been engineered to
enhance their intrinsic tolerogenic potential, either by modify-
ing epitopes to render them more active immunologically, by
combining several self-peptides or by conjugating immunosup-
pressive drugs. Carriage of self-peptides on nanoparticles combined
with cell-targeting molecules and immuno-modulatory drugs has
emerged as a potent platform to re-induce tolerance to self-
components.
More recently, transfer of immune cells has been considered as
potential tolerogenic solutions for many immune-related diseases,
such as RA. For instance, tolerogenic dendritic cell differentiated
ex vivo and loaded with self-peptides was shown to efficiently
reduce disease severity and incidence. Alternatively, the transfer
of autoreactive Tregs, expanded ex vivo or genetically engineered
to express self-antigen-specific chimeric receptors or self-peptides,
has emerged as a potential approach to cure RA.
Although promising results have been obtained in mouse
models, the translation to human pathologies remains highly chal-
lenging since the results of clinical trials often do not match the
6
Joint Bone Spine 88 (2021) 105164
pre-clinical observations. Several reasons might explain this dis-
crepancy.
Firstly, disease-causing immunogenic self-antigens are often
unknown for patients, while many arthritis mouse models rely
on the injection of a given self-peptide (mainly collagen II or PG),
whose pathogenic role in humans remains unclear. In addition,
the appropriate self-peptide(s) for treatment would be different
among patients. To address this issue, libraries of self-peptides
for treatment might become available not only for adapting the
therapy to every patient, but also to restore the tolerance against
several self-peptides at the same time, thus counteracting epi-
tope spreading. Additionally, by selecting organ-specific antigens
that are expressed in the joints for instance, tolerance to unknown
disease-causing self-antigens might also be restored by bystander
effect. Nevertheless, animal models that more closely mimic human
pathologies are required to achieve a deeper understanding of the
pathology and testing for the most appropriate therapeutic strat-
egy. Towards this goal, several mouse models humanised with
either human joint molecules or cells, or with human immune cells
have been developed in the past decades and will certainly become
more widely used in preclinical studies on RA [63].
Secondly, the time course of monitoring of the clinical trials
needs to be carefully considered and standardised, as the beneficial
effects might take several months or years to be detected, since the
return to a state of tolerance is likely not immediate.
Finally, most studies in mice tested prophylactic approaches,
while in humans, the treatments generally start when the disease
is already well established, which limits their efficacy. Previ-
ous or ongoing treatments of patients with anti-inflammatory
drugs may also impact the efficacy. Interestingly, autoantibodies
are often detected years before the onset of the disease, leaving
a room for restoration of tolerance, before the disease reaches
an active phase. Further knowledge on prognostic biomarkers,
disease-causing agents and pathogenesis will obviously help for
the early disease management.
Alternatively, besides self-peptides, other molecules may be
used as disease biomarkers, such as pro-inflammatory cytokines.
For instance, a chimeric inflammation-sensitive promoter induc-
ing the expression of IL-10 in immune cells was shown to strongly
impact arthritis incidence and severity [64]. In addition, with the
expansion of molecular engineering, programmable synthetic sen-
sors can be designed in order to recognise a disease-biomarker
molecule that can be chosen to trigger the expression of a desired
effector in response to sensing [65].
Overall, approaches to achieve antigen-specific tolerance hold
great promises for long-term RA treatment and will surely benefit
from technological progresses, such as gene and cell engineering, in
order to develop clinically safer and more effective strategies in the
near future. Additionally, similar approaches may also be tested for
other autoimmune pathologies and for overcoming organ rejection
after transplant. Conversely, the development of efficient treat-
ments for other autoimmune pathologies would certainly widen
the possibilities for RA treatment as well.
Author contributions
A.P. conceptualised the manuscript, wrote the initial draft and
revised subsequent drafts, F.F and F.-L.C. reviewed and edited the
manuscript prior to submission. All authors have read and agreed
to the published version of the manuscript.
Disclosure of interest
The authors declare that they have no competing interest.
A. Page, F. Fusil and F.-L. Cosset
Acknowledgments
The authors acknowledge the critical reading of this manuscript
by Dr. Alexandre Belot.
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