Single domain antibody-antigen adducts that target Class II MHC induce antigen-specic tolerance

The association of autoimmune diseases with particular allelic variants of Class II MHC (MHCII) products implicates presentation of the offending self-antigen(s) to T cells. Antigen presenting cells are tolerogenic when they encounter antigen under non-inammatory conditions. Manipulation of antigen presentation would therefore be a possible intervention to induce antigen-specic tolerance. We show that, under non-inammatory conditions, systemic administration of a single dose of a nanobody that recognizes MHCII (VHH MHCII) conjugated to the relevant self-antigen affords long-lasting protection against induction of experimental autoimmune encephalitis (EAE), type 1 diabetes (T1D), and rheumatoid arthritis (RA). Co-administration of the VHH MHCII-antigen adduct together with dexamethasone, conjugated to VHH MHCII via a cleavable linker, not only halted progression of established EAE in symptomatic mice but even reverted the severity of EAE, establishing this approach as a potential means of treating autoimmune conditions. Violet trace 2D2 T day infusion, spleens sorted then for


Introduction
Approximately 10% of the human population suffer from an autoimmune condition, with symptoms that range from mild to life-threatening [1]. Current treatments for autoimmune diseases include general immunosuppression, which blunts responses across the entire spectrum of antigens. Various preclinical models of autoimmunity involve administration of a de ned antigen under the appropriate stimulatory conditions to elicit pathology [2]. Engagement of antigen presenting cells (APCs) under in ammatory conditions, e.g., in the presence of adjuvants, elicits a strong response against foreign antigens [3,4]. In contrast, APCs that acquire antigen under non-in ammatory conditions fail to upregulate costimulatory signals and induce tolerance [5,6]. To target APCs under tolerogenic conditions, we developed and characterized alpaca-derived single domain antibody fragments (nanobodies/VHHs) that recognize Class II MHC molecules (VHH MHCII ) [7,8]. These nanobodies lack effector functions and target all MHCIIpositive cells, which include APCs. Their small size ensures e cient tissue penetration and rapid clearance from the circulation of those nanobodies that fail to nd their target. This makes nanobodies ideal vehicles for targeted delivery of payloads of interest, such as antigenic peptides or small molecule drugs [9,10]. We have further established an engineering strategy for nanobodies that enables their sitespeci c modi cation at their C-terminus with the aforementioned payloads [11].
The distribution of a diverse set of APCs over different anatomical sites and their cellular dynamics complicate the identi cation of the relevant tolerogenic APC in vivo. The possible transfer of materials between various sets of APCs is an additional confounding factor. In fact, several distinct types of APCs or their products could all contribute and act in synergy to induce tolerance [12]. Here, we demonstrate the e cacy of using VHHs that target the MHCII-positive cell population as tolerogens. This approach was effective in prevention and treatment of experimental autoimmune encephalitis (EAE), in an accelerated model of type I diabetes in the mouse and in a T cell-mediated arthritis model. To extend this approach to disease interception, we delivered dexamethasone, an immunosuppressive small molecule, to MHCIIpositive cells via VHH MHCII conjugated to dexamethasone through a cleavable linker. We nd that VHH MHCII -peptide adducts in combination with VHH MHCII -dexamethasone are an effective treatment of animals symptomatic for EAE: it halts disease progression in animals with overt signs of disease.

Results
A single dose of VHH MHCII -MOG  provides durable protection against induction of experimental autoimmune encephalomyelitis (EAE). We engineered an alpaca-derived single domain antibody that recognizes a wide range of mouse Class II MHC molecules (MHCII), including I-A b and I-A d (VHH MHCII ), with a sortase recognition motif -LPETGG -to allow its site-speci c ligation (Fig. 1a) to antigenic peptides and small molecules modi ed with (a) suitably exposed glycine residue(s). Puri ed VHH adducts were characterized by LC-MS ( Fig. 1b and Supplementary Fig. 1) to verify identity.
Immunization of C57BL/6 mice with MOG  in the presence of complete Freund's adjuvant (CFA) and pertussis toxin (PTX) elicits experimental autoimmune encephalitis (EAE), a multiple sclerosis-like condition [13]. We hypothesized that prior administration of MOG  , delivered to MHCII+ APCs under non-in ammatory conditions, might interfere with the induction of EAE. We administered 3 doses of 20mg of the VHH MHCII -MOG  adduct intravenously, 7 days prior to induction of disease. This treatment suppressed induction of EAE. Mice that received the identical amount of VHH MHCII conjugated to an irrelevant peptide (VHH MHCII -OVA 323-339 ) or MOG  peptide linked to a VHH of irrelevant speci city (VHH GFP ) invariably developed EAE (Fig. 1c, Supplementary Fig. 2). Even a single injection of 20mg VHH MHCII -MOG 35-55 still achieved full protection ( Fig. 1d and e, Supplementary Fig. 2); a dose used in all subsequent experiments. Flow cytometry of CD4+ lymphocyte in ltrates recovered from the spinal cord of diseased mice at day 15-18 after immunization and of protected mice at day 30 after administration of the MOG 35-55 /CFA/PTX cocktail was consistent with the observed disease scores: diseased mice showed a pronounced in ux of IL-17 and IFNg-producing CD4+ T cells as well as some Foxp3+ CD4+ regulatory T cells (Fig. 1f, Supplementary Fig. 2). H&E and Luxol Fast Blue staining of spinal cord sections from mice that received VHH MHCII -MOG  prior to attempts to induce EAE showed preservation of myelination and less immune cell in ltration (Figs. 1g and 1h).
To explore the durability of protection afforded by VHH MHCII -MOG 35-55 , we injected 20mg of VHH MHCII -MOG 35-55 one or two months prior to administration of the MOG 35-55 /CFA/PTX cocktail. Even then we observed delayed onset, if not complete suppression of EAE (Fig. 1i, Supplementary Fig. 3). In spite of the short circulatory half-life of free VHH MHCII -MOG  , estimated to be < 0.5 hour, VHH MHCII -MOG  confers prolonged protection. Five weeks after the VHH MHCII -MOG  injection, which established protection to a rst exposure to MOG 35-55 /CFA/PTX, we re-challenged mice with a second administration of MOG  /incomplete Freund's adjuvant (IFA) in the presence of PTX. Despite this second challenge, mice, once protected, showed no signs of developing EAE (Fig. 1j, Supplementary Fig. 4). Tolerance evoked by a single dose of VHH MHCII -MOG  , even weeks after its administration, thus provides lasting protection to even a second challenge. Splenic CD11c+ DCs are the APCs associated with induction of antigen-speci c tolerance. To explore possible mechanisms of VHH MHCII -mediated induction of tolerance, we generated VHH MHCII -Alexa 647 ( Supplementary Fig. 5) to follow the biodistribution of VHH MHCII -Alexa647, injected i.v. into MHCII-GFP mice. These mice carry a targeted gene replacement that encodes an I-A b -GFP fusion. It replaces the endogenous I-A b locus and ensures that all Class II MHC+ cells express GFP [14]. At 1.5 hrs after injection, VHH MHCII -Alexa647 is captured by a splenic and circulatory MHCII-GFP+ cell population (Fig. 2a). The uorescent VHH MHCII adducts were captured by B cells and DC subsets, including splenic CD8a+ DCs, CD4-conventional DCs (cDCs), as well as CD4+ cDCs, but not plasmacytoid DCs ( Supplementary Fig. 6).
Intravenous, but not subcutaneous or intraperitoneal injection of VHH MHCII -MOG 35-55 protected against induction of EAE ( Supplementary Fig. 7). This hinted at a role of the spleen or the bloodstream as a site where tolerance induction is initiated. One week after injection of 20mg of VHH MHCII -MOG  i.v. (Fig. 2b,   Supplementary Fig. 8), we harvested their splenocytes and whole blood as a source of donor cells. Mice then received 20 millions unfractionated splenocytes or peripheral blood mononuclear cells (PBMCs) from the VHH MHCII -MOG 35-55 treated animals. One day after cell transfer, we administered MOG  in CFA + PTX to induce EAE. We saw a signi cant reduction in the mean clinical EAE score in mice that received splenocytes from mice treated with VHH MHCII -MOG   (Fig. 2b, Supplementary Fig. 8). We eliminated macrophages and CD8 T cells in vivo by administering the corresponding depleting antibodies: anti-CFS1R antibodies and anti-CD8a antibodies respectively (Fig. 2c, Supplementary Fig. 9) [15]. To deplete DCs, we administered diphtheria toxin (DTX) in CD11c-DTR (diphtheria toxin receptor) mice (Fig. 2c, Supplementary Fig. 9) [16]. To test the possible involvement of B cells, we administered VHH MHCII -MOG 35-55 into mMt-/-mice, which lack mature B cells [17]. Only elimination of CD11c+ DCs reduced the measure of protection provided by VHH MHCII -MOG   (Fig. 2c, Supplementary Fig. 9). We created two VHH-MOG  adducts that presumably target different but overlapping subsets of myeloid cells: we used a VHH directed against CD11b (mostly present on macrophages) and a VHH that recognizes CD11c (mostly present on dendritic cells) (Supplementary Fig. 1) [7]. Only the VHH CD11c - To determine whether delivery by VHH MHCII of more than just the minimal epitope can likewise induce tolerance, we generated VHH MHCII -MOG 17-78 and treated mice 7 days prior to challenge (Fig. 2e).
VHH MHCII -MOG 17-78 likewise protected against induction of EAE (Fig. 2f). This shows that our approach can tackle a condition where the offensive protein but not its minimal epitope(s) is known.
Administration of VHH MHCII -MOG 35-55 elicits a burst of proliferation, followed by attrition, of MOG 35-55speci c CD4 T cells. To investigate the impact of VHH MHCII -MOG 35-55 adducts on T cells of de ned antigen speci city we used 2D2 TCR transgenic mice as a source of monoclonal CD4+ T cells that recognize the I-A b -MOG 35-55 complex [19]. We transferred congenically marked, Violet CellTrace-labeled 2D2 CD45.2+ CD4+ T cells into CD45.1 recipients, followed by injection i.v. of VHH MHCII -peptide adducts one day later. We tracked the number of 2D2 cells in spleen, inguinal lymph nodes (iLNs), and blood for 10 days. In mice receiving VHH MHCII -MOG  , 2D2 CD4+ T cells underwent an initial burst of expansion, followed by contraction on day 5 post-injection, as judged from the absolute number of 2D2 cells recovered from spleen, iLNs, and blood as well as whole body imaging using non-invasive positron emission tomography (PET) for CD4+ cells (Fig. 3a, Supplementary Fig. 12). Such disappearance occurred after several divisions: all of the recovered 2D2 CD4+ T cells were antigen-experienced and had divided, as evident from Violet CellTrace dilution (Fig. 3b). Delivery of an amount of MOG  equimolar to that of the administered VHH MHCII -MOG 35-55 adduct led to division of no more than ~5% of the 2D2 T cells. VHH MHCII -mediated antigen delivery thus clearly enhances its presentation (Fig. 3b).
MOG-speci c 2D2 CD4 T cells upregulate co-inhibitory receptors upon administration of VHH MHCII -MOG  . We next examined the transcriptome of 2D2 T cells in VHH MHCII -MOG 35-55 recipients. We sorted 2D2 CD4 T cells at different divisional stages (Fig. 3b) and performed RNAseq analyses. Injection of VHH MHCII -MOG 35-55 upregulates co-inhibitory receptor transcripts as well as negative regulatory transcription factors. LAG3 transcripts stand out in both magnitude and signi cance (Fig. 3c, d, Supplementary Fig. 13). At the protein level, these 2D2 T cells also showed higher levels of apoptotic and exhaustion markers, such as PD1 and LAG3, but not Tim3, Fas/CD95, or LAP (Fig. 3e, Supplementary Fig.  14) [20]. At day 3 post-injection, 2D2 CD4 T cells in VHH MHCII -MOG 35-55 recipients failed to down-regulate CD62L, while remaining CD44+ ( Supplementary Fig. 14). When we treated LAG3-/-mice with a single dose of VHH MHCII -MOG  and then attempted to induce EAE, protection was lost, albeit with signi cant delay, whereas PD1-/-mice were still tolerized by VHH MHCII -MOG   (Fig. 3f). Deletion of LAG3 in 2D2 TCR transgenic mice has been shown to cause spontaneous EAE [21]. This indicates the importance of LAG3 pathway in this tolerance induction strategy.
Finally, we challenged mice that had received 2D2 T cells with MOG 35-55 /CFA at day 10. The 2D2 T cells in mice that received VHH MHCII -MOG  failed to respond, whereas 2D2 T cells in mice injected with VHH MHCII -OVA 323-339 proliferated robustly (Fig. 3g). This underscores the antigen speci city of tolerance induction by VHH MHCII -MOG  . VHH MHCII -antigen adducts act in an antigen-speci c manner in other models of autoimmunity. We next explored other examples of autoimmunity. For type-1 diabetes (T1D), we used the aggressive BDC2.5 T cell adoptive transfer model, which mimics destruction of β-cells by autoreactive T cells. TCR transgenic CD4 T cells that carry the BDC2.5 T cell receptor recognize pancreatic β cells and can be activated ex vivo with a 10-residue peptide, the mimotope p31. In NOD/SCID mice, such activated BDC2.5 T cells cause hyperglycemia within 8 days after transfer [24]. We conjugated p31 to VHH MHCII ( Supplementary Fig. 1).
Arthritis can be induced in BALB/c recipients by intravenous transfer of ex vivo activated DO11.10 T cells that recognize OVA 323-339 , followed one day later by re-stimulation in vivo with a footpad injection of OVA/CFA emulsion and a challenge 10 days later by injection of heat-aggregated ovalbumin ( Fig. 4c) [25]. Mice were monitored for development of arthritis by measuring paw thickness and by histological assessment at day 7 following a challenge with heat-aggregated ovalbumin. Prior administration of VHH MHCII -OVA 323-339 reduced joint in ammation upon exposure to ovalbumin, whereas VHH MHCII -MOG 35-55 had no effect (Fig. 4c, Supplementary Fig. 17). Mice treated with VHH MHCII -OVA 323-339 showed fewer signs of cartilage destruction (Fig. 4d). Immune cells obtained from popliteal lymph nodes of mice treated with VHH MHCII -OVA 323-339, when stimulated ex vivo with OVA, failed to produce IFNg Combined, these results con rm the ability of VHH MHCII -antigen adducts to reduce the harm in icted by activated, autoreactive CD4 T cells. The underlying mechanism(s) must be conserved across mouse MHC haplotypes. Mice received congenically marked OTI T cells, followed by injection of VHH MHCII -OTI or VHH MHCII -ORF8 (with or without adjuvant) a day later (Fig. 4e). The ORF8 epitope derived from MCMV is recognized by CD8 T cells in H-2 b mice and served as a control [27]. A re-challenge of the recipients with OVA/CFA at day 10 post transfer failed to activate any remaining OTI T cells (Figs. 4e, f). To explore whether B cell responses are similarly affected by administration of VHH MHCII-antigen adducts, we modi ed VHH MHCII with a B cell-speci c OVA-derived epitope (OB1) (Supplementary Fig. 1) [28]. Three consecutive injections of VHH MHCII -OBI into C57BL/6J recipients failed to elicit IgG antibody responses against either intact OVA protein or the OB1 peptide (Figs. 4g, h), whereas mice that received equimolar amounts of free OVA protein readily produced such antibodies.
Co-delivery of VHH MHCII -MOG  and VHH MHCII -dexamethasone improves therapeutic e cacy. We then explored the impact of VHH MHCII -MOG  administration to mice already symptomatic for EAE.
Injection of VHH MHCII -MOG  into mice that had developed an EAE score of 1 (limp tail) halted progression of EAE in 9 out of 16 mice (Fig. 5a, Supplementary Fig. 18). For the remaining 7 out of 16 mice, their overall condition rapidly deteriorated (shivering; reduced motor activity) after injection of VHH MHCII -MOG  , seemingly unrelated to EAE. A cytokine storm elicited by the targeted delivery of antigen into an already in amed environment was responsible (Fig. 5c). The polyclonal nature of the evoked T cell response and the rather super cial clinical scoring system imply heterogeneity in the diseased cohort, which may explain why not all animals that received VHH MHCII -MOG 35-55 responded similarly. We wondered whether it might be possible to co-deliver an immunosuppressive drug to avert a cytokine storm. We delivered the immunosuppressive corticosteroid dexamethasone, attached via a selfimmolating hydrazone linker to VHH MHCII , to Class II MHC+ cells (VHH MHCII -DEX; Fig. 5b, Supplementary   Fig. 19) [29]. Mice that received a combined dose of 20mg VHH MHCII -MOG  and 20mg VHH MHCII -DEX survived and reverted to lower clinical EAE clinical scores, without obvious side effects (Fig. 5d). Improvements in clinical score were mirrored by a reduction in in ltrating CD4 T cells in the spinal cord ( Supplementary Fig. 20). The observed bene t required no more than the equivalent of 0.5mg DEX in the form of the VHH MHCII -DEX adduct. Free DEX, on the other hand, provided protection only when administered at a ~200-fold higher dose of 100mg i.p. (Supplementary Fig. 21). We extended the therapeutic range to animals that had progressed to an EAE score of 2 or 3, all of which responded to coadministration of VHH MHCII -MOG  and VHH MHCII -DEX by an arrest in disease progression, again without side effects. Affected mice even showed a signi cant amelioration in disease score (Figs. 5e, f, Supplementary Fig. 20).

Discussion
Various modes of antigen delivery can induce antigen-speci c tolerance in pre-clinical models of autoimmune disease, with some promise in early-stage clinical trials [30]. Drastic immune 'resetting' by myeloablation, followed by autologous hematopoietic stem cell transplantation, has produced promising results in severely ill patients with myasthenia gravis and multiple sclerosis [31]. Other cell-based therapies include transfusion of modi ed immune cells, including dendritic cells and engineered erythroid cells [32][33][34][35]. Tolerogenic nanoparticles have also been explored as a means of intervention in autoimmunity [36,37]. In addition to curbing in ammation, wholesale immunosuppression is the backstop in the treatment of autoimmunity, a therapy that can increase the risk of infectious disease. While antibiotic treatment can mitigate this drawback at least in part, the search for a more targeted approach to blunt undesirable immune reactions remains a priority.
The induction of antigen-speci c tolerance is a particularly high bar to clear if one considers the presence of pathology and pre-existing autoimmunity at diagnosis. Autoimmune destruction of target cells predates the onset of symptoms that bring the patient to medical attention. Therapy must therefore deal not only with pre-existing autoimmunity but also with the possibility of epitope spreading beyond the initiating insult. Any type of prophylactic treatment will be of limited use unless susceptible populations can be identi ed unambiguously, and then only if the risk of eliciting unwanted side effects of the proposed treatment is acceptably small.
The recent and rather narrow focus on dendritic cells as a key component of cell-based interventions in immunity has overshadowed earlier work in which antigens were targeted to Class II MHC products, expressed on all antigen presenting cells [5,38,39]. This was done through the creation of full-size anti-Class II MHC monoclonal antibodies conjugated to antigens to elicit an immune response. For this reason, we chose to deliver nanobody-autoantigen fusions under non-in ammatory conditions to MHCII+ cells, a strategy that does not obviously differentiate among the various APC subsets, but is e cacious, nonetheless. More importantly, our anti-mouse VHH MHCII does not distinguish between MHCII allotypes.
We showed that it is not essential to deliver the minimal CD4 T cell epitope, but that larger fragments can also lead to tolerogenic antigen processing and presentation (Figs. 2e, f). Depending on the size of the autoantigen, VHH MHCII -antigen adducts can eliminate the need for precise epitope identi cation in a disease or protein replacement setting. This highlights advantages of the approach reported here, successful in the prevention of autoimmunity in three mouse models, over those that employ de ned allotypes of Class II MHC molecules complexed with relevant antigenic epitopes [40]. Ideally, interventions ought to be antigen-speci c and as simple as possible, both from a manufacturing and application perspective. The VHH MHCII adducts described here meet that criterion. We hypothesize that antigen delivery to Class II MHC+ cells cover the relevant tolerogenic APCs and obviates the need of identifying disease-speci c or organ speci c APCs.
In conclusion, a MOG 35-55 -modi ed VHH that recognizes Class II MHC products can protect mice against the induction of EAE. A single injection of 20mg of the VHH MHCII -antigen adduct affords protection that lasts for at least two months. Administration of the same VHH MHCII -MOG  adduct in animals that already show symptoms of EAE (score 1, 2 or 3) halts progression and even allows partial reversal of the severity of the symptoms. Only a subset of animals symptomatic for EAE responded to treatment with VHH MHCII -MOG 35-55 without undesirable side effects, whereas the remainder showed rapid exacerbation attributable to a cytokine storm. An in ammatory environment must already exist in symptomatic animals, such that delivery of the VHH MHCII -MOG  adduct to APCs only adds fuel to the re. Indeed, administration of nanobody-peptide adducts in the presence of anti-CD40 and poly dIdC as adjuvants strongly potentiates antibody responses against them [7]. To overcome this acute response, we codelivered a VHH MHCII -dexamethasone adduct, which rescued survival. In a setting where there is a chronic in ammatory response, administration of the type of nanobody adducts developed here would be possible only if appropriate supporting measures were available, as in the case of the VHH MHCIIdexamethasone adduct.
The pharmacokinetic properties of nanobodies make them particularly attractive for the construction of antibody-drug conjugates (ADCs) [41,42]. Full-sized immunoglobulin-based ADCs continue to circulate for periods up to weeks and release payloads directly into the bloodstream upon hydrolysis of the linkers via which the drugs are attached. This can result in unwanted systemic drug exposure. In contrast, the VHH MHCII -dexamethasone adduct has the desired properties of excellent targeting, as veri ed by noninvasive imaging, short circulatory half-life and ease of modi cation [43]. The cellular targets recognized by VHH MHCII include all MHCII+ cells. Even if the types of APC responsible for induction of tolerance and for provoking a cytokine storm are several and distinct, the MHCII-based targeting approach would cover them. Nanobody-drug adducts have yet to nd the broad range of applications of their full-sized counterparts, but our data show it is an opportunity not to be discounted and pointed to the immense potential of our method.

Methods
Expression of VHHs and endotoxin removal WK6 E. coli containing the plasmid encoding corresponding VHHs were grown to mid-log phase at 37°C in Terri c Broth plus ampicillin and induced with 1 mM IPTG overnight at 30°C. Bacteria were harvested by centrifugation at 5,000g for 15 minutes at 4 °C and then resuspended in 25 mL 1× TES buffer (200 mM Tris, pH 8, 0.65 mM EDTA, 0.5 M sucrose) per liter culture and incubated for 1 hour at 4 °C with agitation. Resuspended cells were then submitted to osmotic shock by 1:4 dilution in 0.25× TES buffer and incubation overnight at 4 °C. The periplasmic fraction was isolated by centrifugation at 5,000g for 30 min at 4 °C and then loaded onto Ni-NTA (Qiagen) in 50 mM Tris, pH 8, 150 mM NaCl, and 10 mM imidazole. Protein was eluted in 50 mM Tris, pH 8, 150 mM NaCl, 500 mM imidazole, and 10% glycerol and then loaded onto a Superdex 75 10/ 300 column in 50 mM Tris, pH 8, 150 mM NaCl, 10% glycerol.
The peak fractions were recovered and rebounded to Ni-NTA to be depleted of LPS (<2 IU/mg). Bound VHHs were washed with 40 column volumes of PBS + 0.1% TritonX-114 and eluted in 2.5 column volumes endotoxin-free PBS (Teknova) with 500 mM imidazole. Imidazole was removed by PD10 column (GE Healthcare), eluting in LPS-free PBS. Recombinant VHH purity was assessed by SDS/PAGE and LC-MS.

Chemical Synthesis of GGG-antigens, GGG-Cy5 and GGG-DEX
The peptides were synthesized on 2-Chlorotrityl resin (ChemImpex) following standard solid phase peptide synthesis (SPPS) protocol or ordered on GenScript. For GGG-Cy5, GGGC (7.0 mg, 24 µmol) was dissolved in DMSO (Sigma Aldrich) (400 µL) and was added to Cyanine 5 maleimide (Lumiprobe) (5.0 mg, 7.8 µmol). The resulting mixture was gently agitated at room temperature until LCMS analysis show no remaining starting material. The ligated product was then puri ed by RP-HPLC and lyophilized. LC

Flow Cytometry Analyses
Cells were harvested from spleen, lymph nodes, or other organs and were dispersed into RPMI1640 through a 40-micron cell strainer using the back of a 1 mL syringe plunger. Cell mixture were subjected to hypotonic lysis (NH 4 Cl) to remove red blood cells, washed twice in FACS buffer (2 mM EDTA and 1% FBS in PBS) and resuspended in FACS buffer containing the corresponding uorescent dye-conjugated antibodies. All staining was carried out at 1:100 dilution and with Fc block for 30 min at 4°C in dark. Samples were washed twice with FACS buffer before further analysis. All ow data were acquired on a FACS Fortessa ow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).
The following is the list of antibodies used in this study: Experimental Autoimmune Encephalomyelitis (EAE) Model in C57BL/6J mice Female C57BL/6 mice (10-12 weeks of age) or other mouse lines with C57BL/6J genetic background were immunized with Hooke kits: an emulsion of MOG  in CFA and PTX in PBS according to the manufacturer's instructions (Hooke laboratories). Mice were scored daily, starting on day 7 postimmunization by an investigator blinded to the experimental treatment of individual mice. Mice were assigned to different experimental treatments randomly and cohoused together to eliminate inter-cage variability. All treatments were carried out on at least 3 mice and in at least two independent experiments, as indicated in the gure legends. All animals were included in the analyses. Clinical score is de ned as follows: 1, limp tail; 2, partial hind leg paralysis; 3, complete hind leg paralysis; 4, complete hind and partial front leg paralysis; and 5, moribund. Easy access to wet food and water was provided for the experimental mice throughout the disease progression. Unless indicated otherwise, for prophylactic treatment, 20mg sortagged VHH-antigens were intravenously administered 7 days prior to induction of EAE. For therapeutic treatment, 20mg VHH MHCII -OVA 323-339 , VHH MHCII -MOG 35 as per the manufacturer's protocol. 500,000 of these 2D2 CD4+ T cells were transferred into CD45.1+ mice. Transfusion of 20mg VHH MHCII -OVA 323-339 , 20mg VHH MHCII -MOG  , equimolar of MOG  peptides, or 100mg MOG  peptides mixed with 25mg anti-CD40 (SouthernBiotech) and 50mg PolyI:C (Sigma) as adjuvant was carried out the day after adoptive transfer. At day 3, 5, and 10, mice were sacri ced and spleens, iLNs, and blood were collected and analyzed by ow cytometry. Some of these 2D2 T cell adoptively transferred mice were also challenged on day 3 or 10 with 100mg MOG  in CFA subcutaneously. Mice were sacri ced 7 or 5 days later as indicated in the respective experimental set up.
Spleens, iLNs, and blood were harvested and analyzed by ow cytometry.

2D2 CD4 T Cell RNAseq
Cells were sorted and lysed in RLT lysis buffer (Qiagen) supplemented with n-mercaptoethanol. RNA was the isolated using a RNeasy Micro kit (Qiagen) according to the manufacturer's protocol. 20ng of RNA were used as input to a modi ed SMART-seq2 protocol. The resulting library was con rmed using a High Sensitivity DNA Chip run on a Bioanalyzer 2100 system (Agilent), followed by library preparation using the Nextera XT kit (Illumina) and custom index primers according to the manufacturer's protocol. Final libraries were quanti ed using a Qubit dsDNA HS Assay kit (Invitrogen) and a High Sensitivity DNA chip run on a Bioanalyzer 2100 system (Agilent). All libraries were sequenced using Nextseq High Output Cartridge kits and a Nextseq 500 sequencer (Illumina). Sequenced libraries were demultiplexed using the bcl2fastq program and the resulting Fastq data were trimmed and cropped with Trimmomatic. Alignment to the mouse mm10 reference genome and gene expression counts were carried out using Kallisto. Principal Component Analyses (PCA) were carried out in R. To test for differential gene expression from our RNA-seq data and differential chromatin accessibility in individual loci, we used the DEseq2 method.
Volcano plot and heatmaps were generated in Python 3.6 using NumPy 1. 5 million cells were adoptively transferred into 9-12-week-old female NOD.SCID mice via retro-orbital injection. Saline, 20mg VHH MHCII -p31, or VHH MHCII -MOG  were infused into the mice a day or 5 days later as indicated. Blood glucose measurements were carried out every other day for 2 weeks and weekly for up to 1-2 months. Mice were considered diabetic when their blood glucose level exceeded 260 mg/dL for two subsequent weeks as measured by using the Active meter (Accu-Chek) (range 20-600 mg/dL) with corresponding Aviva Plus test strips (Accu-Check).
Mice were sacri ced via asphyxiation at the 2-month endpoint or when blood glucose levels exceeded 600 mg/dL for two subsequent weeks. Interferon gamma (IFNγ) production. IFNγ was measured using the Mouse IFN-γ ELISA Set (BD Biosciences, 555138) per manufacturer's protocol.

Repeated Transfusions of VHH MHCII -OB1
OB1 is a 17-mer B cell epitope derived from OVA. C57BL6/J recipient mice were intravenously injected with 20μg VHH MHCII -OB1, equimolar amount of OVA proteins, or PBS at day 0. Subsequent boosts were carried out on day 7 and day 14. Serum samples were collected pre-immunization and 7 days after the last boost. For OVA-speci c and OB1 peptide-speci c ELISA, 96-well plates were coated with 10 μg/mL of OVA or GFP-OB1 proteins in PBS overnight at 4°C and incubated in blocking buffer (0.05% Tween20 + 2% BSA in PBS) before addition before addition of serum samples. Incubation with tested serum was for 3 hours at room temperature. Plates were washed four times with PBS, incubated with goat anti-mouse IgG-HRP (SouthernBiotech) at 1:10,000 in blocking buffer for 1 hour, and developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) liquid substrate reagent (Sigma). The reaction was stopped with 1 N HCl and absorbance was read at 450 nm.

Statistical Methods
All data represented at least two independent experiments. All statistical analyses were performed using Prism 6. Statistical methods used are indicated in the corresponding legend of each gure. Statistically signi cant differences are indicated by asterisks as follows: ***p < 0.001. . We challenged these mice with OTI peptide emulsi ed in CFA on day 10. Spleens, iLNs, and blood were collected 5 days later and analyzed by ow cytometry. f, Splenocytes were cultured for 3 days in complete RPMI supplemented with OT1 peptide. Supernatant was collected to measure production of IFNg by ELISA. g, h, Antibodies against OB1 peptide (g) and OVA protein (h) were measured by ELISA in sera collected from C57BL/6J recipients that received three consecutive injections of saline, VHHMHCII-OBI, or equimolar amounts of free OVA. Data