RES EARCHRESEARCH ARTICLE◥

MULTIPLE SCLEROSIS

A noninflammatory mRNA vaccine for treatment ofexperimental autoimmune encephalomyelitis
Christina Krienke1,2, Laura Kolb1*, Elif Diken1*, Michael Streuber1, Sarah Kirchhoff1, Thomas Bukur1,Özlem Akilli-Öztürk1, Lena M. Kranz3, Hendrik Berger3, Jutta Petschenka1,4, Mustafa Diken1,3,
Sebastian Kreiter1,3, Nir Yogev5,6, Ari Waisman2,5, Katalin Karikó3, Özlem Türeci3,7, Ugur Sahin1,2,3†

A

ntigen-specific tolerization for the treatment of autoimmune diseases may selectively blunt autoimmunity withoutcompromising normal immune function.
In the past decades, various approaches
have been studied, including delivery of autoimmune antigens using DNA, synthetic peptides,
recombinant proteins, coated nanoparticles, or
immunomodulatory cellular therapies [reviewed
in (1)]. However, clinical translation remained
elusive, with largely negative or inconclusive
outcomes in human studies, and only a few
approaches are in early clinical testing. One
impediment is the polyclonal complexity of
autoimmune diseases driven by distinctive,
diverse autoreactive immune cell repertoiresof patients. The interindividual variability requires either personalized treatment tailored
for the autoantigenic immune profiles of thepatients or therapies that mediate bystander
tolerance to suppress both cognate and noncognate autoimmune lymphocytes without
broad immune suppression (2).

TRON – Translational Oncology at the University Medical
Center of the Johannes Gutenberg University gGmbH,
Freiligrathstr. 12, Mainz 55131, Germany. 2Research Center
for Immunotherapy (FZI), University Medical Center at the
Johannes Gutenberg University, Langenbeckstr. 1, Mainz
55131, Germany. 3Biopharmaceutical New Technologies(BioNTech) Corporation, An der Goldgrube 12, Mainz 55131,
Germany. 4Cancer Immunology and Immune Modulation,
Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer
Str. 65, 88397 Biberach an der Riss, Germany. 5Institute for
Molecular Medicine, University Medical Center of theJohannes Gutenberg University, Mainz 55131, Germany.
6
Clinic and Polyclinic for Dermatology and Venereology,
University Hospital Cologne, Kerpenerstr. 62, Cologne 50937,
Germany. 7CI3 – Cluster for Individualized
Immunointervention e.V., Hölderlinstraße 8, 55131 Mainz,
Germany.
1*These authors contributed equally to this work.
†Corresponding author. Email: sahin@uni-mainz.de

Krienke et al., Science 371, 145–153 (2021)

The physiological induction and maintenanceof peripheral tolerance is based on the presentation of self-antigens by lymphoid antigenpresenting cells (APCs) with low-level surface
expression of costimulatory molecules, such as
CD86. We sought to develop a therapeutic approach that would emulate natural mechanismsof immune tolerance. We recently introduced aliposomal formulation for systemic delivery ofmRNA-encoded vaccine antigens (mRNA-LPX)
into lymphoid tissue-resident CD11c+ APCs (3).
mRNA vaccination induces strong type 1
T helper (TH1) cell responses driven by highlevels of interferon-a (IFN-a), released from
APCs upon Toll-like receptor (TLR) signaling(3). Replacement of uridine (U) by incorporation of 1-methylpseudouridine (m1Y) duringin vitro transcription and subsequent removalof double-stranded mRNA contaminants is
known to abrogate TLR7-triggering activity andto reduce inflammatory properties of singlestranded mRNA (4–6). We hypothesized thatthe use of such nucleoside-modified, purified
mRNA (m1Y mRNA) for in vivo delivery of
autoimmune disease target antigens into CD11c+
APCs in a noninflammatory context would enable systemic tolerogenic antigen presentation
in lymphoid tissues.
Noninflammatory delivery of antigen-encoding
m1Y mRNA into the spleen expands
antigen-specific CD4+ regulatory T cells

To test this hypothesis, we engineered
nanoparticle-formulated mRNA-LPX (hereinreferred to as mRNA) consisting of nonimmunogenic (m1Y) or immunogenic (U) mRNA
complexed with liposomes that lack inherent
adjuvant activity. In a first experiment, mRNA
coding for the reporter gene firefly luciferase

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The ability to control autoreactive T cells without inducing systemic immune suppression is the major
goal for treatment of autoimmune diseases. The key challenge is the safe and efficient delivery ofpharmaceutically well-defined antigens in a noninflammatory context. Here, we show that systemic
delivery of nanoparticle-formulated 1 methylpseudouridine-modified messenger RNA (m1Y mRNA)
coding for disease-related autoantigens results in antigen presentation on splenic CD11c+ antigenpresenting cells in the absence of costimulatory signals. In several mouse models of multiple sclerosis,
the disease is suppressed by treatment with such m1Y mRNA. The treatment effect is associated with areduction of effector T cells and the development of regulatory T cell (Treg cell) populations. Notably,
these Treg cells execute strong bystander immunosuppression and thus improve disease induced bycognate and noncognate autoantigens.

(LUC) or saline as control was administeredintravenously into albino C57BL/6 mice, andthe translation and expression of the LUC
protein was assessed.
In line with previous reports, administration of U mRNA led to strong activation of
CD11c+ APCs and lymphocytes, and secretionof high levels of IFN-a (Fig. 1, A to C, and fig.
S1, A and B). By contrast, we did not observe
secretion of IFN-a or other inflammatory cytokines or significant activation of CD11c+
APCs, CD8+ and CD4+ T cells, or natural killer(NK) and B cells in m1Y mRNA–treated mice
(Fig. 1, A to C, and fig. S1, A and B). Notably,translation of LUC was profoundly higher andprolonged in m1Y mRNA–treated animals(Fig. 1, D and E). These findings suggest that
m1Y mRNA is suitable for noninflammatory
delivery of proteins into splenic CD11c+ APCs.
To study the effects of m1Y mRNA in an
autoimmune disease, we chose experimental
autoimmune encephalomyelitis (EAE), a clinically relevant mouse model of multiple sclerosis (MS), in which we previously demonstrated
tolerance induction by selectively expressing
MOG35-55, the epitope of myelin oligodendrocyte glycoprotein, in dendritic cells (DCs) (7).
We assessed the effect of antigen-encoding
m1Y mRNA treatment on T cell expansion.
Naïve Thy1.2+ C57BL/6 mice were immunizedwith MOG35-55 m1Y or U mRNA, and the expansion of both endogenous T cells and adoptively transferred MOG35-55–T cell receptor
transgenic Thy1.1+ CD4+ T cells from 2D2 mice(8) was assessed. Both MOG35-55 –encoding
mRNAs induced proliferation of adoptivelytransferred CD4+ 2D2 T cell, with MOG35-55
m1Y mRNA being superior (Fig. 1F). Similarly,
endogenous MOG35-55–specific CD4+ T cells innaïve mice were expanded by both MOG35-55–
encoding mRNAs (Fig. 1G). However, the functional properties of the T cells induced witheither of these mRNAs differed profoundly.
MOG35-55 m1Y mRNA treatment was capableof expanding or inducing de novo Foxp3+
regulatory T cells (Treg cells) in both wild-type
C57BL/6 and 2D2-Foxp3-eGFP transgenic
mice (Fig. 1H and fig. S2A), whereas overallfrequencies of CD4+ Foxp3+ T cells did notchange (Fig. 1H). CD4+ T cells from vaccinated
2D2 animals inhibited the in vitro proliferationof antigen-specific naïve CD4 T cells in a dosedependent manner. By contrast, CD4+ T cells of
MOG35-55 U mRNA or control-treated mice showed
little to no suppressive activity (fig. S2B).
We studied the cytokine response profiles
upon in vitro antigen restimulation and thephenotypes of expanded T cells in repetitively vaccinated C57BL/6 mice in more detail.
Whereas MOG35-55 U mRNA–expanded T cells
exhibited a functional TH1 effector profile withsecretion of IFN-g, tumor necrosis factor–a(TNFa), interleukin-6 (IL-6), granulocytemacrophage colony-stimulating factor, andRES EARCH | R E S E A R C H A R T I C L E

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Fig. 1. Antigen-encoding m1Y mRNA potently expands antigen-specificCD4+ Treg cells by noninflammatory delivery into the spleen. (A andB) Activation of splenic immune cells 24 hours after (n = 3) and (C) IFN-a serum
levels 6 hours after intravenous injection of LPX-formulated mRNAs and saline(control) in C57BL/6 mice (n = 6). (D and E) Bioluminescence imaging ofalbino C57BL/6 mice (n = 5) after intravenous injection of m1Y or U LUC mRNA.
Representative mice are shown. (F) Frequency and proliferation profiles of
MOG35-55–specific CD4+ T cells isolated from Thy1.1+ 2D2 mice, cell trace
violet (CTV)–labeled, and transferred into naïve Thy1.2+ C57BL/6 recipient mice.
Twenty-four hours after adoptive cell transfer, C57BL/6 mice were treatedwith mRNAs or saline (control). Mice were sacrificed on day 4, and spleens
were analyzed for proliferating CD4+ Thy1.1+ cells (n = 3). (G) Expansion of
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endogenous MOG35-55–specific CD4+ T cells and (H) frequency of splenic
Foxp3+ Treg cells (n = 6) in C57BL/6 mice after treatment (days 0, 3, 7, and10) with mRNA or saline (control) analyzed by MOG35-55–tetramer (tet)staining 3 days after last dosing (n = 4 to 6). (I) CD4+ T cells of mice from(G) tested for IFN-g secretion by enzyme-linked immune absorbent spot
(ELISpot) upon restimulation with MOG35-55 peptide (n = 4 to 5). (J) Phenotypeof tet+ CD4+ T cells of mice from (G). Data were compared by using one-way
analysis of variance (ANOVA) and post hoc Tukey’s test in (A) to (C), (F),and (I) or by unpaired two-tailed Student’s t test in (H) and (J). Mean fluorescenceintensity (MFI) of bulk CD4 T cells of control groups from (H) and (J) are depicted
to show baseline expression levels. Error bars indicate mean ± SD. *P ≤ 0.05;**P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. LLOD, lower limit of detection.
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IL-2 (Fig. 1I and fig. S3), splenic CD4+ T cells
from MOG35-55 m1Y mRNA–treated mice did
not secrete these proinflammatory cytokines,
even when exposed to very high antigen concentrations. The only measurable factors were
low levels of anti-inflammatory and TH2 type–associated cytokines such as IL-10, IL-5, andIL-13 (fig. S3). The T cell exhaustion markers
TIGIT, TIM-3, PD-1, CTLA-4, and LAG-3 werestrongly up-regulated on MOG35-55 m1Y mRNA–
expanded tetramer+ T cells (Fig. 1J).
Exposure to m1Y mRNA does not impair thecapability to mount immune responses

Treatment with antigen-encoding m1Y mRNAameliorates EAE in mice

Next, we studied the tolerogenic potential of
MOG35-55 m1Y mRNA in C57BL/6 micewith MOG35-55–induced EAE. Treatment with
MOG35-55 m1Y mRNA was capable of blocking all clinical signs of EAE in mice (Fig. 3A),
whereas control animals showed the typicalcourse of the disease with rapid monophasic
progression. In mice started on MOG35-55 m1YmRNA treatment when a paralysis of the tailor beginning of the hindlimbs were noted(disease score of 1 to 2 of EAE), further diseaseprogression could be prevented, and motor
Krienke et al., Science 371, 145–153 (2021)

Treatment with m1Y mRNA leads totherapeutically effective bystander tolerance

In another experimental setup, we addressed a
key challenge in human MS, namely that
antigen spread leads to a complex antimyelin
autoreactivity pattern and the specificity of

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autoreactive T cell clones in individual patients, and thus, the potential targets for direct antigen-specific tolerization is unknown.
A clinically viable approach would be to use
bystander tolerance by inducing Treg cells,
which, once activated by their cognate antigen,would suppress T cells against other antigens
in the inflamed tissue.
We evaluated bystander activity in two experimental settings. F1 C57BL/6 x SJL micewith PLP139-151 peptide–induced EAE were
vaccinated with m1Y mRNA encoding either
PLP139-151 (the disease-causing autoantigen),
MOG35-55 (unrelated autoantigen, against which
m1Y mRNA is capable of inducing potent
effector Treg cells), or irrelevant m1Y mRNA
(Fig. 4A and fig. S7C). MOG35-55 m1Y mRNAtreatment showed a dose-dependent therapeutic effect on EAE, similar to the curative
effect mediated by vaccination with PLP139-151
m1Y mRNA, indicating a strong Treg cell–
mediated bystander suppression, also given
the fact that antigen spread has been described
for this particular EAE model (14). Antigenspecific Treg cells were notably expanded andhighly activated, constituting >80% of de novo
expanded MOG 35-55 –specific CD4 + splenic
T cells (Fig. 4, B and C). Teff cell infiltration
into the brain and spinal cord in m1Y mRNA–treated mice was reduced (Fig. 4, D to F), andno signs of demyelination in the spinal cord
were detected (Fig. 4G).
We also investigated a complex EAE model
driven by multiple pathogenic autoreactive
T cell clones against MOG35-55 , PLP139-151,
PLP178-191, MBP84-104, and MOBP15-36, which
could be successfully treated with the mixtureof m1Y mRNAs coding for the corresponding
four disease-inducing autoantigenic epitopes.
m1Y mRNA encoding MOG35-55 were therapeutically almost as effective as those encoding the cocktail of all four disease targets. Thissuggests that even polyclonal autoimmune
disease driven by a broad autoreactive T cell
repertoire can be sufficiently controlled by
m1Y mRNA targeting a strong bystander
tolerance–mediating T cell epitope (fig. S7D).
One potential risk associated with antigenspecific tolerization is the induction of autoantibodies against respective targets, which canexacerbate disease (15). Moreover, nucleosidemodified mRNA is known to be highly immunogenic and to induce high antibody titers
when formulated with immune stimulatory
lipid nanoparticles (16). We therefore analyzed anti–MOG35-55 immunoglobulin G (IgG)
antibody responses in the sera of EAE miceupon m1Y mRNA vaccination. First, we measured anti–MOG35-55 levels in sera of MOG35-55
peptide–induced EAE (C57BL/6 mice), whichwere vaccinated with MOG35-55 m1Y mRNAon days 7 and 10 after EAE induction. Anti–
MOG35-55 IgG levels were not elevated in comparison with those of control animals treated
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CD11c+ APCs of mice first exposed to m1YmRNA and thereafter injected with U mRNA
did not show any impairment in their ability
to respond to this TLR-agonistic stimulus with
up-regulation of costimulatory molecules andIFN-a secretion (Fig. 2, A and B). To investigate whether the induction and expansionof MOG35-55–specific CD4+ Treg cells affects
de novo priming of antigen-specific immune
responses, we exploited two broadly usedmodel systems. First, C57BL/6 mice underwent
prime-boost (days 6 and 13) vaccination with U
mRNA encoding the ovalbumin (OVA) epitope
SIINFEKL and were concurrently exposed to
MOG35-55–encoding m1Y mRNA (days 0, 3,
7, and 10). SIINFEKL-specific CD8+ T cells
were expanded above 40% of total blood CD8+
T cells (Fig. 2C) and displayed properties ofeffector T (Teff) cells such as cognate IFN-g secretion and highly potent and antigen-specifickilling (Fig. 2, D and E), which suggested uncompromised T cell priming and expansion.
In a second experiment, mice were immunizedintramuscularly with a self-amplifying RNA(saRNA) vaccine encoding influenza hemagglutinin (HA) (day 6) concurrent to repeatedtreatment with MOG35-55 m1Y mRNA or controls. Again, the capability of mice to mount a
protective immune response and develop neutralizing antibodies was unimpaired (Fig. 2,
F and G). Overall, both studies demonstratethat MOG35-55 m1Y mRNA–induced antigenspecific CD4+ Treg cells do not suppress functional immune responses against nonmyelin
antigens.

functions were restored (Fig. 3B and fig. S4A).
This included occasional cases of reversion ofparalysis, which was most likely attributable
to an anti-inflammatory effect rather than tissue repair.
Various effects were observed in mice treatedwith antigen-encoding m1Y mRNA comparedwith control animals. In the brain and spinal
cord, the total amount of infiltrating CD4+
T cells, MOG35-55–specific CD4+ T cells andsubsets of CD4+ T cells secreting IFN-g and
IL-17A were considerably lower (Fig. 3, C, D,
and F, and figs. S4B and S5). Demyelinationof the spinal cord was also considerably reduced (Fig. 3E). In the spleen of MOG35-55 m1Y
mRNA–treated animals, we observed an increase of lymphocytes (fig. S5), including
MOG35-55–specific CD4+ T cells with low CD62L,
CCR6, and CCR7 expression, and up-regulationof CD69 (fig. S6). CCR6, CCR7, and CD62L are
critical for access of T cells to the central nervous system (CNS) (9–12), and the transmembrane C-type lectin CD69 is known to promote
lymphocyte retention in the spleen (13).
Next, we analyzed the autoantigen-specificCD4+ T cells in those mice in which the manifestation of EAE was prevented by treatment
with MOG35-55 m1Y mRNA on days 7 and 10.
MOG35-55–specific splenic CD4+ T cells fromtreated animals showed down-regulation of
the activation marker CD44 and strong expression of coinhibitory molecules (Fig. 3G). At
the peak of disease (day 16 after disease induction), CD5, ICOS, LAG-3, PD-1, CTLA-4, TIGIT,
and TIM-3 were up-regulated in tetramer+
splenic CD4+ T cells. Furthermore, we detecteda highly activated Treg cell population (Fig. 3, H
and I) and lower numbers of TH1 and TH17
MOG35-55–specific CD4+ T cells (Fig. 3H). Similarfindings in mice with symptomatic EAE (diseasescore of 1 to 2 at start of treatment) further
confirmed the potent disease-suppressive activity of antigen-encoding m1Y mRNA (fig. S4).
We extended our study of the preventive
and therapeutic effect of m1Y mRNA to other
EAE mouse models. The SJL model is based on
autoreactivity against the PLP139-151 epitope andis characterized by recurring EAE symptoms
resulting in a relapsing-remitting disease, similar to the clinical presentation of MS in patients. Treatment of SJL mice with PLP139-151
m1Y mRNA twice a week starting from day 7
after EAE induction resulted in almost fulldisease control (fig. S7A). Even when the mice
were treated after the first disease peak (starting on day 14 after disease induction), progression of the disease was halted (fig. S7B).

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blood

spleenblood

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spleenFig. 2. Exposure to m1Y mRNA does not impair the capability to mountimmune responses. (A) Activation of splenic CD11c+ APCs 24 hours after(n = 3) and (B) IFN-a serum levels 6 hours after intravenous injection (day 3)of LPX-formulated U mRNA or saline (control) in C57BL/6 mice (n = 6), whichwere pretreated with m1Y mRNA or saline (control) at day 0. MHCII, major
histocompatibility complex II. (C to E) De novo priming of SIINFEKL-specific CD8+
T cells in C57BL/6 mice with prior exposure to MOG35-55 or irrelevant m1Y
mRNA or saline (days 0, 3, 7, and 10) to SIINFEKL U mRNA prime-boost
vaccination (days 6 and 13). Controls only received saline. (C) Frequency of
SIINFEKL-specific CD8+ T cells (OVA257-264 tet) in blood (n = 5 to 7) and spleen(n = 3 to 4). (D) IFN-g secretion was measured by ELISpot upon restimulationof total splenocytes of mice from (C) (n = 3 to 4) with SIINFEKL peptide and(E) in vivo antigen-specific killing of adoptively transferred CTV-labeled andpeptide-loaded splenocytes of naïve mice (n = 4 to 5). For in vivo cytotoxicity
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assays, mice were adoptively transferred on day 18 with 0.5 mM (low) or
5 mM (high) CTV-labeled naïve splenocytes pulsed with peptide (6 mg/ml). Of
these target cells, 1.5 × 106 cells were adoptively transferred into immunized
and control recipients at a ratio of 1:1 (irrelevant HA518-526 peptide–loaded
CTVlow:SIINFEKL peptide–loaded CTVhigh). Recipient splenocytes were recovered
and analyzed by flow cytometry 18 hours after transfer, and antigen-specificlysis was determined as follows: specific lysis (%) = [1 − (percentage of cells
pulsed with SIINFEKL/percentage of cells pulsed with HA) × 100]. (F) Total(n = 5 to 6) and (G) neutralizing (n = 9 to 11) anti-HA IgG in C57BL/6 mice withprior exposure to MOG35-55 or irrelevant m1Y mRNA or saline (days 0, 3, 7,
and 10). Controls only received saline. Protective immune responses were measured
28 days after mice were immunized with influenza HA-saRNA (1 mg intramuscularly)
on day 6. Data were compared by using one-way ANOVA and post hoc Tukey’s
test. Error bars, mean ± SD. DOD, change in optical density; VN, virus neutralizing.
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Fig. 3. Treatment with antigen-encoding m1Y mRNA ameliorates EAE inmice. (A and B) Disease severity in MOG35-55–induced EAE (n = 6 to 8 C57BL/6
mice per group) treated with m1Y mRNA or saline (control) (A) on days 7
and 10 after disease induction or (B) when disease progressed to a score of 1 to
2. (C) Frequency of CD4+ T cell and MOG35-55–specific CD4+ T cells in the
spinal cord of mice treated on days 7 and 10 after disease induction (n = 3 to 4).
Thy1.1+ 2D2 CD4+ T cells were transferred 1 day before EAE induction into
Thy1.2+ recipient mice and analyzed in the target organs on day 16 after
disease induction. (D) Representative CD4 staining in the spinal cord of EAE
mice treated with m1Y mRNAs or saline (control) (n = 3) on days 7 and
10 after disease induction and analyzed at day 16 after EAE induction.
(E) Representative Luxol fast blue (LFB) staining revealing areas of demyelination
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in the spinal cord of mice from (D) (n = 3). (F to I) Frequency of CD4+ IFN-g–
and IL-17A–secreting cells in brain and spinal cord (n = 3 to 4) (F) andflow cytometry analysis of splenic tetramer+ CD4+ T cells (n = 4 to 6) [(G) to(I)] of EAE mice treated with m1Y mRNAs or saline (control) on days 7 and
10 after disease induction and analyzed at day 16 after EAE induction.
Area under the curve (AUC) was used to determine statistical significancethrough one-way ANOVA and Tukey’s multiple comparison test of the different
EAE disease development curves in (A) and (B). Data were compared byusing one-way ANOVA and post hoc Tukey’s test in (F) and (H). Error bars
indicate mean ± SEM in (A) and (B) or mean ± SD in (C), (F), and (H). The scalebar in the upper row of (D) and (E) represents 200 mm and, in the lower rowof (D) and (E), represents 50 mm.
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spleenbrain

spinal cord

spinal cord

Fig. 4. Treatment with m1Y mRNA leads to therapeutically effective
bystander tolerance. (A) Dynamics of EAE in PLP139-151–induced EAE mice(n = 13 to 15 F1 C57BL/6 x SJL mice) upon treatment with MOG35-55 m1Y,
PLP139-151 m1Y, irrelevant m1Y mRNA, or saline (control) twice per week startingon days 7 and 10 after disease induction with 40 mg of m1Y mRNA. (B andC) Expansion of endogenous MOG35-55–specific CD4+ T cells (B) and frequencyof Tetramer+ Foxp3+ Treg cells and analysis of CD69 expression on respective
cell population (C) upon treatment with m1Y mRNA measured in the spleenon day 28 after EAE induction (n = 5). (D) Frequency of CD4+ IFN-g– and IL-17A–
secreting cells upon PLP139-151–peptide restimulation and (E) total cell count of
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brain

spleenlymphocytes in brain and spinal cord of EAE mice treated with different m1Y
mRNAs (n = 4 to 5) and analyzed on day 28 after EAE induction. (F) Representative
CD4 staining in the spinal cord of EAE mice from (A) (n = 2). (G) Representative
LFB staining revealing areas of demyelination in the spinal cord of mice from(A) (n = 2). AUC was used to determine statistical significance through one-way
ANOVA and Tukey’s multiple comparison test of the different EAE diseasedevelopment curves (A). Data were compared by using one-way ANOVA andpost hoc Tukey’s test. Error bars indicate mean ± SEM in (A) or mean ± SD in (C)to (E). The scale bar in the upper row of (F) and (G) represents 200 mm and,in the lower row of (F) and (G), represents 50 mm.
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Fig. 5. Distinct splenic
antigen-specific CD4+
T cell subsets are expanded
in EAE mice treated with
antigen-encoding m1Y
mRNA. (A to G) MOG35-55–
specific CD4+ T cells isolated
from mice with MOG35-55–
induced EAE treated with
m1Y mRNA on days 7
and 10 after disease induction
and analyzed by single-cell
RNA sequencing on day 15
or day 16, respectively.
(A) A two-dimensional
uniform manifold approximation and projection (UMAP)
projection of MOG35-55–
specific CD4+ T cells isolated
from different treatmentgroups (each dot represents
one cell) identified byunsupervised clustering.
(B) UMAP projection of single
cells, color-coded according
to the identified cell subsets.
(C) Frequency of different
cell subsets. (D) and (E)depict a UMAP projection ofclassical Treg cell markers(D) and effector Treg cellmarkers (E). (F) Genes upregulated in an effector Treg
subpopulation upon MOG35-55
m1Y mRNA treatment(adjusted P value < 0.05).
av. exp., average expression.
(G) Genes down-regulated
upon MOG35-55 m1Y mRNAtreatment in TH1, TH17, and
TH1/TH17 cell subsets(adjusted P value < 0.05).
(H and I) MOG35-55–induced
EAE in C57BL/6 mice(n = 8 per group) treatedwith m1Y mRNA on days 7
and 10 after disease induction
in combination with (H),anti–PD-1, and (I) anti–
CTLA-4 blocking antibodies or
isotype controls administered
twice per week. Statisticalsignificance of AUC
differences of EAE diseasedevelopment curves wasassessed by using one-way
ANOVA and Tukey’s multiple
comparison test in (H) and(I). Error bars represent
mean ± SEM in (H) and (I).
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with irrelevant m1Y mRNA or saline (fig. S8A).
This suggests that m1Y mRNA application does
not exacerbate preformed autoantibody responses. Even after repetitive MOG35-55 m1YmRNA challenge (twice per week, 12 times
in total) of F1 C57BL/6 x SJL mice, no anti–
MOG 35-55 IgG antibodies were detected inthose where EAE was induced with PLP139-151–
peptide immunization (fig. S8B).
Distinct antigen-specific CD4+ T cell subsets
are expanded in EAE mice treated with
antigen-encoding m1Y mRNA

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PD-1 and CTLA-4 signaling contribute to the
induction and maintenance of antigen-specifictolerance

Expression of coinhibitory receptors such as
CTLA-4 and PD-1 on effector and Treg cells is a
key mechanism of immune homeostasis. We
therefore assessed the mechanistic contribution of these pathways to antigen-specific tolerance mediated by m1Y mRNA. Treatment of
EAE mice with MOG35-55 m1Y mRNA combined with anti–PD-1 or anti–CTLA-4 antibody on days 7 and 10 after EAE induction
aggravated the disease in control groups inaccordance with the known unleashing effectof checkpoint blockade on autoreactive T cells(29). Both CTLA-4 and PD-1 blockade almost
completely abolished the EAE-protective effectof MOG35-55 m1Y mRNA (Fig. 5, H and I). Flowcytometry and single-cell RNA sequencing revealed almost exclusive and high expression of
Ctla4 by MOG35-55–specific CD4+ Treg cells (fig.
S16, A and B), which supports a role of thispopulation for mediating tolerance. The effectof PD-1 blockade may be driven by two inde-

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pendent mechanisms, one being invigorationof preexistent Teff cells and the other being theinhibition of the de novo induction of antigenspecific Treg cells, which also depends cruciallyon PD-1 signaling (30). These findings suggestthat disease-mediating T cells are suppressed
but not deleted in mice treated with antigenencoding m1Y mRNA and that both PD-1 andCTLA-4 signaling critically contribute to the
induction and maintenance of antigen-specifictolerance.
Discussion

Our study describes nanoparticulate delivery of
nucleoside-modified autoantigen-encodingmRNA into lymphoid CD11c+ APCs as a therapeutic approach for antigen-specific tolerization. We show, at single-cell resolution, thegeneration of different antigen-specific CD4+
T cell subpopulations with distinct functional
states.
Selective delivery of autoantigens into CD11c+
APCs resident in lymphoid tissues exploits ahighly effective natural mechanism for induction and maintenance of peripheral tolerance.
The presentation of autoantigens in a noninflammatory context leads to expansion of
antigen-specific CTLA-4+, ICOS+, IL-10+, andFoxp3+ effector Treg cells (31) that not only
suppress antigen-specific autoreactive Teff cells
but also exert bystander immunosuppression,
thereby enabling disease control even in acomplex, polyclonal model of autoimmunity.
Bystander activity of Treg cells has been associated with noncognate mechanisms depending on cell-cell interaction, such as secretion of
suppressive factors, e.g., IL-10 and transforming
growth factor–b (TGF-b) (32). Thus, one would
expect that MOG-specific Treg cells activatedupon reexposure to their cognate antigen in the
CNS would suppress immune responses that
occur at that very location for a defined periodof time. Because of its temporo-spatial nature,
Treg cell activity exerts tissue-specific immune
regulation rather than pan-immune suppression (33). This is in line with prior studies indicating that T cell tolerance to tissue-restrictedself-antigens is actively mediated by antigenspecific Treg cells rather than deletion (34).
With the presented approach, key challenges
for clinical translation of antigen-based treatment of autoimmune diseases can be addressed.
Both the nucleoside-modified, purified mRNAand the liposomal nanoparticle formulation
are pharmaceutically well-defined clinical-stage
compounds and are currently being explored inhuman trials for various disease indications(35). The repetitive administration of m1YmRNA is not compromised by induction of
autoantigen-specific antibody responses, whichusually causes safety-limiting constraints forother applications. Production of mRNA pharmaceuticals is fast and cost-efficient, and virtually any autoantigen can be encoded by
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Next, we characterized the tolerized T cells
by subjecting splenic tetramer+ CD4+ T cells
from animals treated with MOG35-55 m1YmRNA or irrelevant m1Y mRNA to single-cell
RNA sequencing (table S1). Clustering analysis
revealed eight distinct antigen-specific CD4+
T cell subsets in EAE mice (Fig. 5, A and B, andfig. S9). The CD4+ T cell identity of all subsets
was confirmed with canonical T cell markers(fig. S10). We used sets of genes characterizing
different functional T cell subphenotypes forfurther analysis (figs. S11 and S12). We identified two distinct populations sharing common Treg cell markers (Foxp3, Ikzf2, and Ctla4).
One of these subpopulations displayed typicaleffector Treg cell markers (Klrg1, Il10, Gzmb,
Lag3, and Icos), whereas the other subset were
central Treg cells as defined by markers Selland Bcl2 and low expression of the common
effector-associated molecules (17, 18) (figs. S11,
A and B, and S13A). We also identified TH1(Ifng and Tbx21), TH17 (Il17a/f and Tmem176a/b), and TH1/TH17 cells with combined expression of TH1 and TH17 markers (Csf2, Tbx21,
Ifng, and Il17a). Moreover, we found exhausted
antigen-specific T cells (Lag3 and Cd160),
T follicular helper (TFH) cells (Il4, Il21, and Cxcr5),
and a T cell cluster with a memory phenotype(Ccr7high and Tcf7high) (figs. S11, C to H, andS13, B and C).
The relative frequencies of these antigenspecific CD4+ T cell subpopulations changed
in EAE mice treated with antigen-encoding
m1Y mRNA. Most notably, in animals treatedwith MOG35-55 m1Y mRNA, the effector Treg
cells constituted the largest cluster by frequency and cell count (Fig. 5C). The TH1, TH17,
and TH1/TH17 Teff cell subpopulations werestrongly reduced in comparison with those ofcontrol animals treated with irrelevant m1Y
mRNA, whereas exhausted T cell and central
Treg cell subpopulations were of similar size
in both groups (Fig. 5C). We also found substantial differences in the expression levelsof functionally relevant genes and activation
markers. Common Treg cell markers such as
Foxp3, Ikzf2, and Ctla4 were present in Treg
cell subpopulations of both treatment groups(Fig. 5D), whereas transcripts characteristic
for effector Treg cells and associated with Treg
cell suppressive function, such as Klrg1, Il10,

Gzmb, Lag3, and Icos, were enriched in MOG35-55
m1Y mRNA–treated EAE mice and almost completely absent in those treated with irrelevant
m1Y mRNA (Fig. 5, E and F). Likewise, thetranscript profiles of the TH1, TH17, and TH1/
TH17 Teff cell clusters differed considerably between the two treatment groups (Fig. 5G andfig. S14A). The most prominent examples ofdifferentially regulated genes were those involved in differentiation (Anxa1, Ppp4c, andMid1) (19–21), migration (Ccr2, Itgb1, Wdr26,
and Rap1b) (22–26) or cytokine production of
Teff cells (Bhlhe40) (27). In accordance with aprevious study (28), we detected mainly downregulation of genes associated with cell cycle
and cell division in the mice treated with
MOG35-55 m1Y mRNA (fig. S14B). The numberof genes up-regulated in TH1, TH17, and TH1/
TH17 Teff cell subpopulations from MOG35-55
m1Y mRNA–treated mice was small (fig. S14A).
To visualize the relationships between theidentified major cell populations, we performed
single-cell trajectory analysis (fig. S15). Eachstate represents subpopulation structures ofclosely related transitory cellular states. T cells
from mice treated with irrelevant m1Y mRNAappeared mainly as fully differentiated cells instate 5, representing TH1, TH17, and TH1/TH17
Teff cells (fig. S15, B, D, and F). By contrast,
T cells from the MOG35-55 m1Y mRNA–treatedgroup were in state 1 (memory) or mainly
differentiated in state 2, representing effector
Treg cells and TFH cells (fig. S15, B, C, and F). In
sum, these findings indicate that m1Y mRNAtreatment rather than deleting autoreactive
T cells tips the immunological balance in favorof suppression of disease promoting MOG35-55–
specific TH1, TH17, and TH1/TH17 Teff cells by
expanding effector Treg cells.

RES EARCH | R E S E A R C H A R T I C L E

mRNA. Thus, tailoring the treatment for the
disease-causing antigens of individual patients
is conceivable, similar to that which has beensuccessfully executed in the setting of personalized cancer vaccines (36, 37). Combination of
m1Y mRNAs encoding either multiple personalized autoantigens or autoantigens that confer bystander tolerance may enable control ofeven complex autoimmune diseases.
RE FE RENCES AND N OT ES

Krienke et al., Science 371, 145–153 (2021)

T. Regen for experimental advice, and L. Giese for supporting single-cell
RNA sequencing. Furthermore, we thank the NIH Tetramer Core
Facility for providing the MOG35-55 MHC class II tetramer. Funding:This work has been supported by grants from the Immunology
Research Center (FZI) Mainz (FZI-TRP 2014-12 to U.S. and N.Y.) andthe Deutsche Forschungsgemeinschaft DFG (SFB/CRC-TR 128 toA.W.). Author contributions: U.S. was responsible for conception
and experimental strategy of the study. Planning and analysisof the experiments were done by C.K., L.M.K., M.D., S.Kr., H.B., andJ.P., supported by N.Y. and A.W. C.K. and S.Ki. performed
immunological experiments. Processing and analysis of scRNAseq data was done by L.K., E.D., and M.S., supported by T.B. Ö.A.-Ö.
performed IHC experiments. C.K., Ö.T., and U.S. interpreted thedata and drafted the manuscript. L.K., E.D., M.S., M.D., K.K., andA.W. supported writing of the manuscript. All authors edited
and approved the final manuscript. Competing interests: L.M.K.,
H.B., K.K., Ö.T., and U.S. are employees at BioNTech SE (Mainz,
Germany). M.D. and S.Kr. work as consultants for BioNTech
SE (Mainz, Germany). C.K., J.P., L.M.K., M.D., S.Kr., K.K., and U.S.
are inventors on patents and patent applications related to this study.
Ö.T. and U.S. are stock owner and management board members of
BioNTech SE (Mainz, Germany). All other authors declare no competinginterests. Data and materials availability: Correspondence andrequest for materials should be addressed to U.S.

ACKN OWLED GMEN TS

science.sciencemag.org/content/371/6525/145/suppl/DC1
Materials and Methods
Figs. S1 to S16
Tables S1 and S2References (38–52)

We thank V. Ames, K. Zwadlo, A. Plaschke, I. Beulshausen,
E. Petscherskich, E. Daniel, R. Roth, B. Jesionek, M. Brkic, A. Selmi,
M. Baiersdörfer, and S. Berl for technical assistance; S. Witzel,
B. Tillmann, S. Wurzel, Z. Yildiz, and N. Blaumeuser for cloning of
constructs; S. Fesser, K. Tillmann, J. Beckerle, E. Heintz, and
C. Golletz for mRNA production; and P. Guna, A.-L. Popa, and
H. Haas for providing liposomes. Additional support was provided
by S. Attig and A. Hohberger for cell sorting. Moreover, we acknowledge
F. Vascotto for support and scientific discussion for the manuscript,

8 January 2021

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View/request a protocol for this paper from Bio-protocol.
13 June 2019; resubmitted 27 April 2020
Accepted 17 November 2020
10.1126/science.aay3638

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A noninflammatory mRNA vaccine for treatment of experimental autoimmune
encephalomyelitis
Christina Krienke, Laura Kolb, Elif Diken, Michael Streuber, Sarah Kirchhoff, Thomas Bukur, Özlem Akilli-Öztürk, Lena M.
Kranz, Hendrik Berger, Jutta Petschenka, Mustafa Diken, Sebastian Kreiter, Nir Yogev, Ari Waisman, Katalin Karikó, Özlem
Türeci and Ugur Sahin

Science 371 (6525), 145-153.
DOI: 10.1126/science.aay3638

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Precision therapy for immune tolerance
Autoimmune diseases, such as multiple sclerosis (MS), result from a breach of immunological self-tolerance andtissue damage by autoreactive T lymphocytes. Current treatments can cause systemic immune suppression and side
effects such as increased risk of infections. Krienke et al. designed a messenger RNA vaccine strategy that lacks
adjuvant activity and delivers MS autoantigens into lymphoid dendritic cells. This approach expands a distinct type ofantigen-specific effector regulatory T cell that suppresses autoreactivity against targeted autoantigens and promotes
bystander suppression of autoreactive T cells against other myelin-specific autoantigens. In mouse models of MS, thevaccine delayed the onset and reduced the severity of established disease without showing overt symptoms of general
immune suppression.
Science, this issue p. 145