Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment

Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide–MHCII complexes.

T he general approach for generation of monoclonal antibodies (MAb) reactive to a defined protein antigen has been well documented since the original report in 1975 by Drs. Kohler and Milstein 1 . The utility and broad use of MAbs in biological systems earned Kohler and Milstein the Nobel Prize for medicine in 1984 (ref. 2). In this report we describe a novel methodology to specifically and reliably generate MAbs that target peptide in the context of MHCII, which has only occurred a few times since 1975 (refs 3-9).
To generate a MAb using the traditional approach, mice are immunized, the responding B cells are isolated, fused to myeloma cells with hypoxanthine-aminopterin-thymidine (HAT)-based selection, screened and sub-cloned to isolate monoclonal hybridomas 2 . Screening requires the examination of hundreds or even thousands of clones for one MAb, creating a major bottleneck. This approach typically yields o1-5% hybridomas specific for a protein target antigen causing a prominent hurdle, both in time and resources 4 . However, this method is not specifically designed to generate peptide:MHCII (p:MHCII) reactive MAbs, and B-cell tolerance against self MHC adds to the difficulty. To overcome this, we developed a novel methodology to generate MAb against a specific p:MHCII complex. B-cell clones specific for the antigen of interest are enriched immediately before myeloma fusion, thus significantly reducing the screening required. The basis for this methodology centers on having a stable p:MHCII monomeric protein linked to biotin as the B-cell antigenic target. This approach has several advantages. First, immunization with p:MHCII complexes induces a B-cell response specific for that peptide in the context of MHCII. Second, use of antigen-specific tetramers allows us to pre-screen immunized mice to confirm the expansion of p:MHCII-specific B cells. Third, it offers the ability to enrich for antigen-specific B cells 10 while discarding B-cell clones responding to unrelated antigens. Specifically, the utility of a site-directed protein biotinylation allows for the enrichment of B cells reactive to the target protein/peptide by generating a tetrameric antigen, thus increasing the avidity of B cells for antigen and enabling the capture and enrichment of antigen-specific B cells 10,11 . This results in a significant time and cost saving as fewer colonies are required for screening, and a higher percentage of selected hybridomas produce MAb against p:MHCII. Finally, this enrichment approach could be used for any MAb protein target including peptides and haptens, not just p:MHCII [12][13][14] .

Results
Generation of p:MHCII MAb. The workflow for this methodology and the necessary steps for p:MHCII MAb generation are illustrated in Fig. 1. Generation and validation of p:MHCII MAb can be completed in o8 weeks. We were interested to develop a reagent to block T-cell receptor (TCR) recognition of a diabetesrelevant peptide [14][15][16] . We initially developed antibodies against p63 peptide in the context of IA g7 MHCII molecule, given that p63-activated BDC2.5 CD4 þ T cells mediate accelerated autoimmune diabetes when transferred into wild-type nonobese diabetic (NOD) hosts [17][18][19] . We isolated splenocytes from five p:MHCII (p63:IA g7 ) immunized BALB/c mice and magnetically enriched for antigen-specific B cells using PEconjugated p63:IA g7 tetramers followed by anti-PE magnetic beads 10 . To validate successful priming and expansion, we analyzed the phenotype of p:MHCII-specific B cells in naive mice compared to day 7 post immunization ( Fig. 2a). Antigenspecific B cells were identified from immunized mice by p:MHCII-PE tetramer excluding those that bound to streptavidin (SA)-phycoerythrin (PE) or SA-allophycocyanin (APC) using SA-PE-AF647 or SA-APC-DyLight 755, compared to a decoy p:MHCII-APC reagent (Fig. 2a). Three distinct subsets of antigen-specific B cells (p:MHCII specific, MHCII specific and decoy p:MHCII specific) 10,20 were evaluated for GL7 and intracellular Ig expression associated with mature germinal center B cells. Phenotypic analysis demonstrates the p:MHCII-PE þ population is enriched for mature germinal center B cells (GL7 þ and intracellular Ig À ) demonstrating successful priming and T-cell help (Fig. 2a). We verified the enrichment approach at day 3 post antigen boost, before hybridoma fusion. Magnetic enrichment resulted in an increase to 11.1% of the B cells staining positive for p63:IA g7 -PE tetramer, and phenotypic markers demonstrating the presence of germinal center B cells within this population (Fig. 2b). The enriched fraction contained 2.1 Â 10 7 cells, which was 23-fold reduced compared with the starting population. These cells were subsequently fused with SP2/0 myeloma cells and plated onto ten, 100 mm plates containing semi-solid media under HAT selection. Fourteen days after plating, 190 colonies were picked and screened by enzyme-linked immunosorbent assay (ELISA). Without enrichment, we would have required 50 plates to screen 5 Â 10 8 cells. We predict that these 50 plates would have contained at least 5,000 colonies, most of which could not have been selected or screened for further analysis due to time and reagent constraints. Thus, enrichment allowed us to screen every visible colony, and saved significant time and reagents.
Screening and in vitro validation of p:MHCII MAb. After expansion of each colony, secreted antibody in the culture supernatant was assessed for binding to p63:IA g7 compared to decoy InsB 10-23: IA g7 by ELISA. Thirty-two of the 190 colonies produced antibodies that bound to both, p63:IA g7 and InsB 10-23 : IA g7 , indicating specificity for an IA g7 epitope (Table 1). In contrast, 11 hybridomas produced antibodies that bound only p63:IA g7 (34.4% success rate for p:MHC or 5.8% overall), suggesting the desired specificity for this peptide bound to IA g7 (Table 1). Figure 3a illustrates 20 clones, 10 that reacted to both p63:IA g7 and InsB 10-23 :IA g7 (bottom), and 10 that are unique for p63:IA g7 (top). We further characterized these 10 clones that uniquely bound p63:IA g7 for TCR blocking ability to limit in vitro antigen-specific T-cell proliferation (Fig. 3b). Splenocytes were isolated from TCR transgenic BDC2.5 mice, labelled with carboxyfluorescin succinimidyl ester (CFSE) and cultured with p63 peptide in the presence or absence of hybridoma supernatant for 4 days. BDC2.5 splenocytes incubated with peptide alone resulted in 87.5% CD4 þ T cells proliferating, while T cells incubated with peptide plus hybridoma A1 limited BDC2.5 T-cell proliferation to 56% (Fig. 3b). The remaining nine hybridomas screened had varying degrees of inhibition (Fig. 3b). We then used an isotypespecific ELISA to determine that A1 antibody was IgG1. A largescale purification was next performed to obtain purified MAb from hybridoma A1 (named FS1). Using the FS1 MAb (anti-p63:IA g7 ) we performed an in vitro dose-response assay and demonstrated 80.5% specific reduction in proliferation with 1.72 mM FS1 MAb (Fig. 3c). In contrast, the FS1 MAb only reduced BDC2.5 T-cell proliferation to another BDC2.5 mimetope (p31) by 5.6% compared with an isotype control (Fig. 3c). p63-activated BDC2.5 T cells demonstrated 99.85% reduction in IFNg production when cultured with 1.72 mM of FS1 MAb, compared with an isotype control (Fig. 3c). Importantly, IFNg production by p31-activated BDC2.5 T cells was not altered (Fig. 3c). We noted a similar trend with IL-17A (Fig. 3c). Taken together, these findings illustrate the specificity of the FS1 MAb as p31 differs from p63 by two amino acids at positions P-1 and P1 of the MHCII binding pocket 21 . As an extension of specific binding, splenocytes from NOD mice were p63 peptide-pulsed and stained with labelled FS1 MAb illustrating CD8a þ cDCs (dendritic cells) and B220 þ B cells stained positive for p63 peptide but not ovalbumin peptide (OVA 141-160 ) control (Fig. 3d). The uniform histogram shift suggests a large portion of the DCs and B cells stained with varying levels of FS1 MAb demonstrating peptide presentation in vitro (Fig. 3d). Importantly, CD4 þ and CD8 þ T cells did not stain positive for the FS1 MAb (Fig. 3d). Immunostaining was next performed to demonstrate peptide binding to MHCII in vivo. NOD mice were injected with p63 or OVA peptide in the footpad and 1.5 h later popliteal lymph node cells were stained with FS1 antibody to identify p63-loaded antigen-presenting cells (Fig. 3e). Both DCs and B cells had significantly increased FS1 MAb staining in response to p63 peptide-pulsed compared to OVA peptide (P ¼ 0.005 and P ¼ 0.008, respectively), while T cells showed no specific staining (Fig. 3e).
Using this novel methodology, we also generated an antibody specific for the peptide 2W 12,22 bound to IA b (named W6). Using this reagent, we validated in vitro antigen loading and presentation using bone marrow-derived dendritic cells that were pulsed with green fluorescent protein (GFP)-linked 2W peptide. Results in Figure 4a demonstrate, 47% of the GFP-positive cells were W6 MAb (anti-2W:IA b ) positive and were mostly CD11c þ CD11b þ double-positive cells. We next   20 . Germinal center B cells (GL7 þ and Intracellular Ig À ) and plasma cells (GL7 À and intracellular Ig þ ) were then identified from the various B-cell populations binding these distinct tetramer reagents to demonstrate successful priming. After 28 days, mice were boosted with a second immunization of p63:IA g7 monomeric protein intravenously. Three days following the immune boost, we preformed magnetic B-cell enrichment for splenic B cells binding to the p63:IA g7 tetramer:PE reagent as described in materials and methods. Enriched cells are fused with myeloma fusion partners, expanded and screened for in vitro and in vivo validation.   validated in vivo antigen loading and presentation. C57BL/6 mice were immunized intradermally with either ovalbumin protein or 2W-GFP. At 24 h post injection, MHCII þ antigen-presenting cells from the draining lymph node had increased W6 MAb reactive populations (15%) compared with 1% of controls ( Fig. 4b). In a separate in vivo model, we used the W6 MAb to identify antigen-presenting cells immunized with 2W peptide and two different adjuvants. C57BL/6 mice were immunized with 2W-GFP and either 5 0 -cytosine-phosphate-guanine-3 0 (CpG) or double-mutant heat-labile toxin (dmLT) 23 . Twenty-four hours  In vitro antibody staining on antigen-presenting cells following peptide pulse with p63 or OVA 141-160 using purified clone FS1 MAb to detect p63 loaded cDCs (CD8a þ CD11c þ MHCII þ , CD3e À , F4/80 À ), and B cells (B220 þ ,MHCII þ ,CD11c À ,CD3e À ,F4/80 À ) but not CD4 þ or CD8 þ T cells (CD3e þ , CD11c À , CD11b À , B220 À , F4/80 À ) compared with no p63 peptide negative control. Data are representative from three independent experiments. (e) In vivo staining of antigen-presenting cells with FS1 MAb following footpad immunization. NOD mice received p63 or OVA 141-160 peptide and 1.5 h following injection the popliteal lymph node was collected and stained for antigen-specific presentation using biotinylated FS1 MAb. FS1 MAb staining was detected on DCs and B cells, but not T cells using fluorochrome-linked streptavidin. Statistical significance was calculated using a two-tailed Student's t-test. Data are representative from two independent experiments with 2-4 mice per group.
later, draining lymph nodes were assayed by flow cytometry for antigen-specific presentation using the W6 antibody. Shown in Fig. 4c, the W6 MAb identified 27% of the DCs containing GFP compared with 3% in the isotype control group and B70% these cells were CD11b þ CD11c þ CD19 À dendritic cells. These results demonstrate that W6 MAb can identify different subsets of antigen-presenting cells in vivo.
In vivo functional validation of p:MHCII MAb. Next, we sought to determine whether MAbs directed against p:MHCII could prevent TCR recognition in vivo to limit T-cell proliferation. NOD mice were challenged with p63 peptide plus lipopolysaccharide (LPS) with FS1 MAb or Y-Ae 3 (anti-Ea:IA b ) as a negative control. Four days post challenge we measured a significant reduction in antigen-specific T-cell expansion with FS1 MAb administration (Fig. 5a). Using dual fluorochrome tetramer staining and flow cytometry, we detected 30-fold expansion of p63 specific T cells stimulated with p63 þ LPS þ Y-Ae MAb control, compared with only a twofold expansion with p63 þ LPS þ FS1 MAb over naive mice (Fig. 5a). In addition to decreased expansion, we also determined that the FS1 MAb decreased activation and cell cycle progression (Fig. 5a). Next, we used the FS1 MAb in vivo to prevent antigen-specific tolerance, resulting in rapid autoimmune diabetes 19,24 . Specifically, we transferred activated BDC2.5 T cells after 4 days of in vitro stimulation into wild-type NOD pre-diabetic recipients followed by injection of ethylene-carbodiimide (ECDI)-fixed p63 peptide-coupled cells (p63cc) to induce tolerance 14,16 and either control or FS1 MAb and monitored the mice for diabetes. P63cc completely prevented diabetes induction in 100% of the mice, while control bovine serum albumin-coupled cell-treated mice developed severe diabetes (Fig. 5b). Mice given p63cc and FS1 MAb developed diabetes, indicating the FS1 MAb prevented the induction of antigenspecific tolerance in vivo. The W6 MAb was next evaluated for its ability to block 2W-specific in vivo expansion of antigen-specific T cells in response to an acute or chronic bacterial infection. C57BL/6 mice were administered W6 MAb and infected with Listeria monocytogenes expressing 2W 25 . Seven days later the number of activated 2W-specific cells was decreased by 146-fold in response to W6 MAb compared with no antibody control (Fig. 6a). To test in vivo 2W-specific CD4 þ T-cell responses and their contribution to bacterial clearance in a chronic infection, wild type 129 S1 mice were infected with 10 8 Salmonella Typhimurium expressing 2W peptide 26,27 . The mice received a single dose of W6 blocking antibody 14 days post infection and were sacrificed at day 35 to evaluate antigen-specific T-cell proliferation and colony forming units. Infected mice treated with the W6 MAb had significantly lower 2W-specific CD4 þ T cells, higher bacterial burden, and did not clear the Salmonella infection (Fig. 6b,c). These data highlight the critical importance of a single antigen-specific T-cell population and the blocking ability of the W6 MAb to prevent in vivo pathogen clearance.
To compare the affinity of the FS1 and W6 MAb with previously published reagents, we performed a direct side-by-side comparison with known IA g7 or IA b specific antibodies. The results are shown in Table 2, and illustrate that the FS1 MAb (anti-p63:IA g7 ) has 100-fold higher affinity (1.7 Â 10 À 11 ) than the 10-2.16 MAb 28 (anti-IA g7 ) (2.9 Â 10 À 9 K D (M)). The W6 MAb (anti-2W:IA b ) had an affinity comparable to known IA b antibodies (Y3P 29 , Y-Ae 3 and AF6-120.1 (ref. 30)). These results suggest that the two MAb generated had comparable or higher affinity than conventional approaches used to develop MAb.

Discussion
Using this methodology we have generated hybridomas producing six novel anti-peptide:MHCII MAb, and for two of these presented here, we demonstrate the high affinity for antigen and biological capacity to limit TCR engagement, prevent subsequent T-cell activation, label antigen-presenting cells, and in vivo use to prevent tolerance induction and bacterial pathogen clearance. We have demonstrated that this novel methodology is highly efficient due to pre-screening and enrichment, saving both time and resources (Table 1). This approach will be useful to generate additional p:MHCII-targeted MAbs as very few exist. The most well-known is the Y-Ae antibody (anti-Ea:IA b ), which recognizes Ea 52-68 bound to IA b MHCII molecules, and was used to understand central tolerance and alloreactive antigen presentation 3,31,32 . The efficiency of Y-Ae generation has not been described in the literature 3 , however, two more recent reports address this issue. The generation of MAbs specific for two different peptides of hen egg lysozyme (HEL), specifically two HEL 46-61 :IA k (clones B6G and C4H) MAb were identified by screening 500 clones 8 . An additional clone specifically recognizing HEL 116-129: IA k (D8H) was identified by screening 500 different colonies. However, these clones were found to also weakly stain cells expressing IA k in a HEL-independent manner 8 . In a separate report, Dadaglio et al. 9 generated a clone specific for HEL 48-62 :IA k (Aw3.18) by screening 1,000 colonies. Thus, the efficiency of generating a MAb clone using conventional approaches has been reported to range from 1:250 to 1:1,000 or , control treatment or untreated naïve mice. P63:IA g7 tetramer PE and APC double postive cells from the spleen were enriched and gated on lymphocyte size, singlets, live cells, B220 À , CD11c À , CD11b À , CD3e þ and CD4 þ . Statistical significance was calculated using a two-tailed Student's t-test. Data are representative from two independent experiments with 2-3 mice per group. (b) In vivo blockade of antigen-specific tolerance using FS1 MAb to prevent ethylene-carbodiimide antigen-fixed-coupled cell tolerance. NOD mice received activated BDC2.5 TCR transgenic CD4 þ T cells to induce diabetes. Ten mice per group received either p63-coupled cells or BSA-coupled cells. Five mice per treatment received FS1 MAb or control. Mice were followed daily for diabetes after day 3 by blood glucose measurements. Data are representative from two independent experiments.
(0.1-0.4%). In Table 1, we report a range of 1:18-1:115 using the novel methodology described here (0.9-13.5%). Taken together, our success rate is 2.25 to 33.75fold higher than traditional approaches resulting in a higher efficiency. The fact that fewer clones are required for positive identification also results in a significant advantage of lower costs and fewer hours required for hybridoma screening.
More recently, an antibody against insulin B peptide 9-23 in IA g7 (anti-InsB 9-23 :IA g7 ) was generated and shown to delay diabetes in NOD mice 4 . In the current study, the FS1 MAb was generated against a diabetes-relevant peptide in the context of IA g7 . Here, we demonstrate successful blockade of antigen-specific tolerance using FS1 MAb causing rapid diabetes (Fig. 5b). Future work using FS1 MAb will characterize the role for polyclonal or monoclonal T cells during spontaneous autoimmune diabetes and tolerance 16,33 . The W6 reagent was generated to understand T-cell responses during both homoeostasis and bacterial pathogenesis, as the 2W peptide can be engineered into a pathogen of interest. Future work with this reagent will characterize cells presenting antigen in response to bacterial infections or immunization with different adjuvants.
One limitation to this approach centers on possessing knowledge of the antigen target, availability of p:MHC monomers for immunization and tetramers for B-cell enrichment. With advances of our understanding of peptide binding to both MHCI and MHCII, enhanced algorithms can be developed to better predict peptide register binding leading to greater numbers of p:MHC monomers 6,34,35 . This, combined with high throughput generation of p:MHCII monomers by peptide exchange 36 would alleviate the current limitations of this approach. The generation of MAb targeting mouse p:MHC class I (MHCI) could also be performed using this methodology. In addition, human MAb targets against peptides in HLA class I or II would also be amenable to this approach, thus offering great potential for human immunotherapy in the context of autoimmunity, tumour immunity or allograft rejection. The ability to specifically block antigen presentation to T or B cells during bacterial or viral pathogenesis could provide mechanistic insight for immunity and regulation, particularly with respect to persistent infection. Finally, the use of MAb specific for p:MHC allows the study of specific subsets of antigen-presenting cells during every aspect of immune recognition ranging from immune homoeostasis to defining novel roles for multiple subsets of antigen-presenting cells responding to vaccination and infection.
We anticipate the need for immunotherapeutics in the form of MAbs directed against p:MHCII will increase as new pathways and novel antigens are identified during disease. These targets could include not only foreign antigens, but also allogeneic antigens during transplantation, tumour neo-antigens, self-proteins targeted during autoimmunity, or bacterial and viral antigens from infected cells. As these targets are identified, there will be a great need to limit antigen-specific T-cell responses, and the methodology described here offers a more efficient approach over conventional protocols.

Methods
Mice. Female NOD mice (6-8 weeks of age) were purchased from Taconic. Female C57BL/6 (6-8 weeks of age), female 129 S1 (6-8 weeks of age) and female BALB/c  mice (8-10 weeks of age) were purchased from the Jackson Laboratory. Female (6-week-old) NOD.BDC2.5 Thy1.1 transgenic mice were bred under specific pathogen-free, barrier facility at the University of Minnesota. Animals were housed under specific pathogen-free, barrier facility in accordance with NIH guidelines. All animal procedures were approved by the University of Minnesota or Tulane Institutional Animal Care and Use Committee.
Briefly, peptide:IA g7 or peptide:IA b molecules were expressed in Drosophila S2 cells using the DES Drosophila Expression System kit (Invitrogen). The S2 cells were co-transfected using calcium phosphate, with plasmids encoding the alpha chain, the peptide-linked beta chain, BirA ligase and a blasticidin resistance gene at a molar ratio of 9:9:9:1 for IA b and IA g7 . Transfected cells were selected in blasticidin-containing Schneider's Drosophila medium (Invitrogen) with 10% fetal bovine serum (FBS), 100 U ml À 1 penicillin/streptomycin (Gibco) and 20 mg ml À 1 gentamycin (Invitrogen) for 1 week at 28°C, passaged into serum-free media containing 25 mg ml À 1 blasticidin (Invivogen), and scaled to 0.5 l cultures in 2 l shaker flasks maintained at 150 r.p.m. When cell densities reached 5 Â 10 6 ml À 1 , monomer expression was induced by the addition of 0.8 mM copper sulphate. Peptide:IA b or IA g7 heterodimers were purified from supernatants 8 days later by immobilized metal ion affinity chromatography using a His-Bind purification kit (Novagen-EMD Biosciences) and eluted using 1M imidazole. The biotinylated pMHCII heterodimers in the eluate were then affinity purified using Monomeric Avidin UltraLink (Pierce). Bound peptide:IA b or IA g7 molecules were eluted with 2 mM biotin in PBS and excess-free biotin was removed by centrifugation and four washes with 12 ml PBS using a 30 KD cut-off Amicon Ultra-15 filter (Millipore). Tetramers were produced by incubating p:MHCII monomers with streptavidin-APC (Prozyme # PJ27S) or streptavidin-PE (Prozyme #PJRS27) at a 4:1 molar ratio. For the detection of SA and fluorochrome specific B cells, AF647 was conjugated to SA-PE (Prozyme) for 60 min at room temperature using an antibody labelling kit (ThermoFisher Scientific) and free AF647 was removed by centrifugation in a 30 KD molecular weight cut-off filter. The concentration was then adjusted to 1 mM PE based on the absorbance at 565 nm using a nandrop spectrophotometer (ThermoFisher Scientific). Similarly, SA-APC (Prozyme) was conjugated to DyLight 755 using an antibody labelting kit (ThermoFisher Scientific) and the concentration was adjusted to 1 mM APC based on the absorbance at 650 nm.
Antigen-specific B-cell enrichment and phenotyping. BALB/c mice were immunized with 50 mg of pMHCII emulsified in complete Freund's adjuvant (CFA, Sigma) subcutaneously in the base of the tail. Seven days post immunization single-cell suspensions from spleen and pooled lymph nodes (inguinal, brachial, cervical and axillary) were prepared by forcing the tissue through a 100 mm cell strainer using the plunger end of a 1 ml syringe, washed with RPMI containing 2% FBS and resuspended in 100 ml Fc block (2.4G2, 0.05% sodium azide). The cells were next incubated with 5 nM SA-PE-AF647, and SA-APC-DyLight755 for 10 min at 25°C, followed by peptide:MHCII-conjugated PE and APC tetramers at Hybridoma selection and specificity screening. Proliferation and cytokine production. Cells were isolated from spleen and peripheral lymph nodes of NOD.BDC2.5 transgenic mice, the red blood cells were lysed by Tris-buffered ammonium chloride, and the cells were labelled with CFSE (ThermoFisher Scientific). Labelled cells were resuspended in complete DMEM media at a final concentration of 4 Â 10 6 cells ml À 1 and p63 or p31 peptide was added to a final concentration of 0.05 mM. Purified monoclonal antibody or 50 ml of hybridoma supernatant was added to each well containing 200 ml of cells in a 96-well plate, and incubated for 4 days at 37°C with 5% CO 2 . Cells were collected, resuspended in 2.4G2 Fc block for 10 min at 4°C, and stained at 1:100 dilution for 30 min at 4°C with antibodies against CD4-BV510 (RM4-5, BD Biosciences), CD8a-BV650 (53-6.7, BD Biosciences), CD3e-PerCp-Cy5.5 (KT4, BD Biosciences), B220-ef450 (RA3-6B2, eBioscience), CD11c-ef450 (N418, eBioscience), CD11b-ef450 (M1/70, eBioscience), TCR Vb4-PE (KT4, BD Biosciences) and dead cells were gated out using a viability ghost red dye (Tonbo), and run on a LSRII Fortessa X-20 flow cytometer (Becton Dickinson) and analyzed using FlowJo software (v10). For antibody dose-response curves, cells were isolated from NOD.BDC2.5 transgenic mice, labelled with CFSE as described above and cultured with p31 or p63 (0.05 mM) with varying doses of FS1 MAb or isotype control (BioXcell). After a 4-day incubation at 37°C with 5% CO 2 , cells were collected for the flow cytometry, while supernatants were analyzed using ProcartaPlex Assay kit EPX170-26087-901 (eBioscience).
In vivo blockade of T-cell activation and tolerance. The NOD mice were administered 50 mg acetylated-p63 peptide with either 250 mg FS1 or Y-Ae MAb in PBS containing 2 mg LPS (Sigma) by i.v. injection in the tail vein. Four days post injection splenocytes were isolated and red blood cells lysed as above. Cells were stained with both APC and PE-conjugated p63:IA g7 tetramers followed by magnetic enrichment of double tetramer positive cells as previously described 14,37 .
Infections. C57BL/6 mice were injected intravenously with 10 7 actA-deficient Listeria monocytogenes expressing 2W protein 25 , and on the same day injected intravenously with 500 mg of W6 (anti-2W:IA b ) blocking antibody. Seven days later, splenocytes were isolated and stained as above and magnetically enriched for 2W:IA b -PE tetramer. Wild type 129S1 mice were infected with Salmonella Typhimurium expressing 2W, and 14 days following infection mice were treated intravenously with 500 mg of blocking W6 MAb. Thirty-five days post infection, the spleen was removed and made into a single cell suspension and 10% was plated for bacterial CFU with the addition of 0.1% Triton (ref. 27). The remaining cell suspension was used for enrichment of 2W specific cells as described above. Cells were run on a LSRII Fortessa X-20 flow cytometer (Becton Dickinson) and analyzed using FlowJo software (v10).
Footpad immunization, antigen-presenting cell isolation and staining. The NOD hind limb footpads were injected with 100 ml total volume containing 200 mg of p63 or OVA 141-160 peptide in PBS. One and half hours later the popliteal lymph nodes were removed, minced and digested in RPMI containing 2% FCS, collagenase D (40 U ml À 1 ) and DNase I (250 mg ml À 1 ) for 30 min at 4°C. Cells were then washed with Hanks balanced salt solution containing 5 mM EDTA and 2% FCS, centrifuged and stained with surface antibodies and analyzed by flow cytometry as described above using the FS1-AF488 antibody.
Ear pinna immunization, antigen-presenting cell isolation and staining. C57BL/6 mice were immunized intradermally in the ear pinna with 10 mg either Ovalbumin (OVA) or 2W-GFP plus 1 mg dmLT (a gift from J. Clements) 23 or 10 mg CpG (Sigma). After 24 h, the cervical lymph nodes were removed, dissociated using a 100-mm mesh and mechanically disrupted, and digested with 300 Mandl U ml À 1 Collagenase D (Roche Applied Sciences) for 30 min at 37°C in 1 Â PBS þ 2% FBS. Cells were then washed 1 Â PBS þ 2% FBS, centrifuged and Fc receptors were blocked in 100 ml 2.4G2 hybridoma supernatant containing 2% rat and mouse serum for 10 min at room temperature. For surface staining, 1 mg of biotinylated W6 antibody was added to the cells and incubated on ice for 45 min, washed with PBS þ 2% FCS, followed by staining for 30 min at 4°C with antibodies CD11c-PerCp-Cy5. 5