Development and Characterization of a Preclinical Model for the Evaluation of CD205-Mediated Antigen Delivery Therapeutics in Type 1 Diabetes Downloaded from Jennifer Schloss, Riyasat Ali, Jeffrey Babad, Ignacio Guerrero-Ros, Jillamika Pongsachai, Li-Zhen He, Tibor Keler and Teresa P. DiLorenzo

ImmunoHorizons 2019, 3 (6) 236-253

doi: https://doi.org/10.4049/immunohorizons.1900014 http://www.immunohorizons.org/ http://www.immunohorizons.org/content/3/6/236 This information is current as of September 27, 2021.

Supplementary http://www.immunohorizons.org/content/suppl/2019/06/26/3.6.236.DCSupp Material lemental References This article cites 66 articles, 26 of which you can access for free at: http://www.immunohorizons.org/content/3/6/236.full#ref-list-1 by guest on September 27, 2021 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://www.immunohorizons.org/alerts

ImmunoHorizons is an open access journal published by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. ISSN 2573-7732. RESEARCH ARTICLE

Clinical and Translational Immunology

Development and Characterization of a Preclinical Model for the Evaluation of CD205-Mediated Antigen Delivery Therapeutics in Type 1 Diabetes

Jennifer Schloss,* Riyasat Ali,* Jeffrey Babad,* Ignacio Guerrero-Ros,† Jillamika Pongsachai,* Li-Zhen He,‡ Tibor Keler,‡ Downloaded from and Teresa P. DiLorenzo*,§,{,k *Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; †Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461; ‡Celldex Therapeutics Inc., Hampton, NJ 08827; §Division of Endocrinology, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461; {Einstein–Mount Sinai Diabetes Research Center, Albert Einstein College of Medicine, Bronx, NY 10461; and kThe Fleischer Institute for Diabetes and Metabolism, Albert Einstein College of Medicine, Bronx, NY 10461 http://www.immunohorizons.org/

ABSTRACT Dendritic cells (DCs) are crucial for the production of adaptive immune responses to disease-causing microbes. However, in the steady state (i.e., in the absence of an infection or when Ags are experimentally delivered without a DC-activating adjuvant), DCs present Ags to T cells in a tolerogenic manner and are important for the establishment of peripheral tolerance. Delivery of islet Ags to DCs using Ag-linked Abs to the DC endocytic receptor CD205 has shown promise in the NOD mouse model of type 1 diabetes (T1D). It is important to note, however, that all myeloid DCs express CD205 in humans, whereas in mice, only one of the classical DC + subsets does (classical DC1; CD8a in spleen). Thus, the evaluation of CD205-targeted treatments in mice will likely not accurately by guest on September 27, 2021 predict the results observed in humans. To overcome this challenge, we have developed and characterized a novel NOD mouse model in which all myeloid DCs transgenically express human CD205 (hCD205). This NOD.hCD205 strain displays a similar T1D incidence profile to standard NOD mice. The presence of the transgene does not alter DC development, phenotype, or function. Importantly, the DCs are able to process and present Ags delivered via hCD205. Because Ags taken up via hCD205 can be presented on both class I and class II MHC, both CD4+ and CD8+ T cells can be modulated. As both T cell subsets are important for T1D pathogenesis, NOD.hCD205 mice represent a unique, patient-relevant tool for the development and optimization of DC-directed T1D therapies. ImmunoHorizons, 2019, 3: 236–253.

Received for publication March 4, 2019. Accepted for publication June 7, 2019. Address correspondence and reprint requests to: Dr. Teresa P. DiLorenzo, Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Forchheimer Building, Room 403, Bronx, NY 10461. E-mail address: [email protected]. ORCIDs: 0000-0001-6739-0902 (J.S.); 0000-0003-4022-276X (R.A.); 0000-0002-2586-1371 (J.B.); 0000-0003-2294-9092 (I.G.-R.); 0000-0001-8206-761X (L.-Z.H.); 0000- 0003-4337-3598 (T.P.D.). This work was supported by National Institutes of Health R01 DK064315, R01 DK094327, R03 AI119225, R01 AI123730, and R01 DK120420 (to T.P.D.), F30 DK103368 (to J.S.), T32 GM007288 (which supported J.S.), P30 CA013330 (to the Cancer Center of the Albert Einstein College of Medicine), P30 DK020541 (to the Einstein-Mount Sinai Diabetes Research Center), the American Diabetes Association (1-16-IBS-069 to T.P.D.), and by grants from the Iacocca Family Foundation and the Diabetes Action Research and Education Foundation (to T.P.D.). J.P. was supported by the Diversity Student Summer Research Opportunity Program of the Albert Einstein College of Medicine. T.P.D. is the Diane Belfer, Cypres & Endelson Families Faculty Scholar in Diabetes Research. Abbreviations used in this article: BMDC, bone marrow–derived DC; cDC, classical DC; CHO, Chinese hamster ovary; DC, dendritic cell; hCD205, human CD205; hIns, human proinsulin; Ins2, murine proinsulin 2; mCD205, murine CD205; MHC II, class II MHC; MoDC, monocyte-derived DC; pDC, plasmacytoid DC; RPMI-10, RPMI 1640 containing 10% FBS, 50 U/ml penicillin and 50 mg/ml streptomycin, 1 mM sodium pyruvate, and 13 nonessential amino acids; T1D, type 1 diabetes. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY-NC-ND 4.0 Unported license. Copyright © 2019 The Authors

236 https://doi.org/10.4049/immunohorizons.1900014

ImmunoHorizons is published by The American Association of Immunologists, Inc. ImmunoHorizons PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES 237

INTRODUCTION only CD205 must be targeted in humans. An unmet need for progress in this area is a preclinical model for the development and Type 1 diabetes (T1D) is characterized by T cell–mediated de- optimization of such a strategy. To meet this need, we have struction of the pancreatic islet b cells (1). Because T1D involves developed and extensively characterized an NOD mouse model inappropriate lymphocyte targeting of the b cells, immune having transgenic expression of hCD205 under the control of the modulatory therapies represent an attractive means of treatment. DC-specific CD11c promoter. As the hCD205 transgene is ex- This is in contrast to merely treating the disease with insulin pressed by both cDC1 and cDC2, the expression of this endocytic replacement, which, although necessary, is unable to stop disease receptor more closely mirrors the situation found in humans. We progression (2). Immune modulatory treatments that have been find that the transgenic mice have a similar T1D disease profile to attempted include the targeting of CD3, CD20, IL-2, or CD80 and standard NOD mice. They retain typical APC subset levels and CD86 (3). These therapies have met with varying clinical success. maturation status for NOD mice, and their DCs remain functional, At best, the response to these short-term treatments was not in addition to being able to process and present Ag taken up via durable, despite initial positive outcomes, and patient disease hCD205. NOD mice with transgenic expression of hCD205 thus returned (3). It is possible that, as T1D is a chronic disease, long- represent a useful model for the preclinical testing of DC-directed term treatment with these reagents would be more successful, but therapies for T1D. Downloaded from this also exposes patients to the complications inherent in immune modulatory therapies. Targeting only T cells specificforb cell Ags is a more attractive option, as it represents a strategy to eliminate MATERIALS AND METHODS or suppress the disease-causing T cells while leaving immune responses to pathogens and cancers intact (4). Using b cell Ags Production of Ag-linked anti-hCD205 Abs http://www.immunohorizons.org/ themselves as treatments is predicated, in part, on the ability of The human anti-hCD205 Ab 3G9 was previously obtained by dendritic cells (DCs) to present Ags in a tolerogenic manner in the immunization of human Ig-transgenic mice with hCD205 (18), absence of a DC-activating adjuvant, leading to T cell tolerance (5, which bears77% aa sequence identity to mCD205. To produce 3G9 6). However, clinical trials where b cellAgsorepitopeswere linked to either OVA, murine proinsulin 2 (Ins2), human proin- delivered to patients have also met with only moderate success (4). sulin (hIns), the mimotope peptide AL [YQLENYCAL; recognized One reason for this may be that previous attempts have delivered by the NOD-derived CD8+ islet T cell clone AI4 (19)], or the HIV- these Ags nonspecifically rather than targeting them directly derived peptide SL9 (SLYNTVATL), each was cloned in frame to DCs. with the C terminus of the 3G9 H chain in a plasmid also encoding Delivery of Ags to DCs in murine models of autoimmunity has the 3G9 L chain. OVA, AL, and SL9 were preceded by a linker shown promise (7–12) and is feasible because of the presence of encoding DMAKKETVWRLEEFGR, derived from the MHC II by guest on September 27, 2021 a number of DC-specific endocytic receptors, including CD205 sequence, whereas Ins2 and hIns were preceded by the sequence

(DEC-205; lymphocyte Ag 75) and DCIR2 (13). These markers, for the flexible linker (GGGGS)3 to allow for proper proinsulin respectively, help define the murine classical DC (cDC) subsets folding (20). Insertion of the proper Ag was assessed by restriction cDC1 and cDC2 (14). Treatment of DCs with Ag-linked Abs against digest analysis and sequencing of the construct. The Abs were these receptors results in the uptake and degradation of the Ag (15). produced by transient transfection of 293T cells using a calcium We have previously shown that delivery of specific epitopes via phosphate transfection kit (Invitrogen). Briefly, 293T cells were murine CD205 (mCD205), expressed only by the mouse cDC1 seeded at 7.5 3 106 cells/150 mm tissue culture dish in 20 ml subset, can lead to induction of CD8+ T cell tolerance toward those DMEM (Life Technologies) containing 10% low IgG FBS (Life epitopes in the NOD mouse model of T1D (8, 9). Others have Technologies), antibiotic/antimycotic (5 U/ml penicillin, 5 mg/ml shownthatdeliveryofAgviaDCIR2,expressedbythemouse streptomycin, and 0.25 mg/ml amphotericin B; Life Technologies), cDC2 subset, results in CD4+ T cell tolerance in NOD mice and and 1 mM sodium pyruvate (Life Technologies) and incubated at delayed T1D onset in an adoptive transfer model (11). Both DC 37°C overnight. The following day, the cells were transfected subsets are, thus, capable of inducing T cell tolerance. This is following the protocol included in the transfection kit with 60 mg important, as the different DC types are proposed to be somewhat of a 3G9 construct and 5 mg of pAdVAntage vector (Promega) per specialized for presentation to different T cell subsets, with cDC1 plate. The pAdVantage vector enhances transient protein expres- (CD8a+ in spleen) optimally presenting to CD8+ TcellsandcDC2 sion by inhibiting the activity of the dsRNA-activated inhibitor (CD8a2)toCD4+ T cells (15). DAI. The transfection solution was added dropwise, and cells were Although humans possess DCs with similar phenotypes to the incubated at 37°C overnight. Cells were then washed with 10 ml of murine subsets, they are not exactly parallel (16). In particular, in serum-free DMEM and incubated for 4–6 d at 37°C in 30 ml of humans, CD205 is expressed by all myeloid DCs, not just the cDC1 DMEM containing antibiotic/antimycotic and 1% Nutridoma subset (17). Because Ags taken up via human CD205 (hCD205) can (Roche). To remove debris, the supernatant was centrifuged at be optimally presented on both class I and class II MHC (MHC II) 486 3 g for 5 min and sequentially passed through 0.45-, 0.22-, and (15), DCs treated in this manner can impact both CD4+ and CD8+ 0.1-mm filters. The supernatant was passed through a Protein G T cells, both of which are important for T1D pathogenesis (1). column (GE Healthcare), and the Ab was eluted with 0.1 M glycine Therefore, to efficiently modulate both T cell subsets, potentially HCl (pH 2.7). Eluted Ab was concentrated using a 30-kDa Amicon

https://doi.org/10.4049/immunohorizons.1900014 238 PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES ImmunoHorizons

Ultra centrifugal filter (EMD Millipore), and three buffer exchanges homozygosity for markers of NODorigindelineatingknownIdd were performed with PBS (pH 7.2). Ab concentration was determined loci (22). The presence of the hCD205 transgene was determined by using a Bradford assay (Bio-Rad) with bovine g-globulin (Bio-Rad) as PCR of tail-tip DNA using the following primer pair: 59-TGGAA the standard, and purity was assessed by SDS-PAGE. GAGACATGGAGAAACCT-39 and 59-TCTCAGGCCAGTCCA GAAGTA-39. The transgene was maintained in a hemizygous Production of Ag-linked anti-mCD205 Abs state. Rag1-deficient OT-I TCR-transgenic C57BL/6 mice were Ins2 was cloned in frame at the C terminus of the H chain of anti- provided by F. Macian (Albert Einstein College of Medicine). mCD205 (NLDC-145), which had been previously modified to C57BL/6 mice transgenically expressing the OT-II TCR were contain murine Ig k and IgG1 constant regions with mutations obtained from The Jackson Laboratory (B6.Cg-Tg[TcraTcrb] that interfere with Fc receptor binding (21). The flexible linker 425Cbn/J; stock no. 004194). NOD mice with transgenic + (GGGGS)3 was inserted in between the Ab and proinsulin to allow expression of the TCRs from the NOD-derived islet CD8 T cell for proper proinsulin folding (20); this Ab was designated anti- clones 8.3 and AI4, denoted NOD.8.3 and NOD.AI4, respectively, mCD205/Ins2. Anti-mCD205/OVA (15) and anti-mCD205/NRP- were maintained in-house and have been described (23, 24). V7 (8) have been described. The Ag-linked anti-mCD205 Abs were produced by transient transfection of 293T cells with calcium Preparation of bone marrow–derived DCs Downloaded from phosphate and purified as described above for the Ag-linked 3G9 Bone marrow cells were harvested from the femur and tibia of Abs, except that for each 150-mm plate to be transfected, 30 mgof NOD and NOD.hCD205 mice and incubated for 3 min in ACK DNA encoding the Ab H chain and 30 mg of DNA encoding the lysing buffer (Lonza). Cells were cultured in RPMI 1640 (Life Ab L chain were used along with 5 mgofpAdVantagevector Technologies) containing 10% FBS, 50 U/ml penicillin and (Promega). 50 mg/ml streptomycin (Life Technologies), 1 mM sodium pyruvate http://www.immunohorizons.org/ (Life Technologies), and 13 nonessential amino acids (Life Assessment of Ag-linked 3G9 binding to hCD205 Technologies) (RPMI-10) supplemented with 10 ng/ml GM-CSF Chinese hamster ovary (CHO), CHO/mCD205 (18), and CHO/ (PeproTech) and 10 ng/ml IL-4 (PeproTech) for 7 d. hCD205 cells (18) were incubated with 3G9/AL (for the experi- ments depicted in Supplemental Fig. 1), 3G9/OVA, 3G9/Ins2, or Splenocyte preparation

3G9/hIns, washed once with PBS containing 1% FBS (HyClone) Mice were sacrificed using CO2 asphyxiation, followed by cervical and 0.1% (w/v) sodium azide, and then incubated with a 1:100 dislocation. Spleens were harvested and placed in ice-cold Ca2+/ 2+ dilution of PE-labeled donkey anti-human IgG (H+L) F(ab9)2 Mg -free HBSS (Life Technologies) supplemented with 40 U/ml (Jackson ImmunoResearch). Samples were washed twice, in- collagenase D (Roche). Collagenase-containing medium was cubated in 1 mg/ml DAPI (Sigma-Aldrich) for 15 min on ice, and injected into each spleen; spleens were then teased into small by guest on September 27, 2021 filtered through a 35-mm cell strainer prior to data collection. Data pieces and digested for 25 min at 37°C. The collagenase was were collected on a BD LSRII flow cytometer with five lasers (355, inactivated by the addition of 10 mM EDTA (Sigma-Aldrich) for 405, 488, 561, and 640 nm) and analyzed using FlowJo software 5 min at 37°C. Samples were then passed through a 40-mm cell (version 8.8.6). strainer, and remaining pieces of spleen were crushed. Samples were washed with 5 ml of RPMI-10 and centrifuged at 486 3 g for Assessment of Ag-linked anti-mCD205 binding to mCD205 5 min. RBCs were lysed in ACK lysing buffer for 4 min at room CHO/mCD205 cells were incubated with anti-mCD205/OVA temperature andwashed with RPMI-10.The resultant splenocytes or anti-mCD205/Ins2, followed by the allophycocyanin-labeled were centrifuged and washed twice with PBS. Prior to the final secondary Ab anti-mouse IgG1 (BD Biosciences). For specific detec- wash, splenocytes were passed through a 40-mm cell strainer. The tion of the proinsulin moiety of anti-mCD205/Ins2, the detection splenocytes were then counted and suspended in PBS. reagents used were 5 mg/ml biotinylated anti-proinsulin (R&D Sys- + tems) followed by 5 mg/ml streptavidin–allophycocyanin (BD Bio- CD11c DC isolation sciences). After washing, cells were resuspended in 5 mg/ml Splenocytes were washed once with autoMACS buffer (Miltenyi DAPI (Sigma-Aldrich) before proceeding to data acquisition. Flow Biotec), counted, and suspended in 40 mlofautoMACSbuffer/107 cytometry data were acquired using a BD FACScan or LSRII and cells and 5 ml of anti-CD11c microbeads (Miltenyi Biotec). Cells analyzed using FlowJo software (Tree Star). were incubated for 30 min at 4°C, washed with 10 ml of autoMACS buffer, and passed through an MS column (Miltenyi Biotec). Mice Following removal from the magnet, the CD11c+ DCs were eluted C57BL/6 mice transgenically expressing hCD205 (C57BL/ from the column, suspended in RPMI-10, and counted. 6.hCD205) (18) were obtained from R. Steinman (The Rockefeller + 2 University). The hCD205 transgene is under the control of a CD8a and CD8a DC isolation by magnetic separation synthetic CD11c promoter (GenBank accession number: DQ658851) Splenocytes were washed once with autoMACS buffer (Miltenyi containing both C57BL/6 and 129 sequences. To generate NOD mice Biotec) and counted. CD8a+ DCs were obtained using a CD8a+ DC with transgenic expression of hCD205, C57BL/6.hCD205 were isolation kit (Miltenyi Biotec). Briefly,splenocytesweredepleted backcrossed for 12 generations with NOD mice and fixed to of T cells, B cells, and NK cells. The remaining cells were labeled

https://doi.org/10.4049/immunohorizons.1900014 ImmunoHorizons PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES 239 with anti-CD8a+ conjugated to magnetic beads, and the cells were (Autoimmun Diagnostika), and the results were analyzed using passed through an MS column. Following removal from the GraphPad Prism 7 software. The stimulation index was deter- magnet, the CD8a+ DCs were eluted from the column. The flow- mined by dividing the number of spots per well by the number of through fraction contained cells that had been depleted of T cells, spots in the negative control wells. B cells, NK cells, and CD8a+ DCs. These cells were labeled with anti-CD11c microbeads (Miltenyi Biotec) and passed through an Analysis of hCD205 expression by APC populations MS column (MiltenyiBiotec). Followingremoval from the magnet, Splenocytes were added to a 96-well V-bottom plate, with 2 3 106 the CD8a2 DCs were eluted from the column, suspended in cells per well. The plate was centrifuged at 486 3 g for 5 min, and RPMI-10, and counted. cells were washed one time in PBS (pH 7.2) containing 2% FBS. This buffer was used for all subsequent washing and dilution steps. + + CD4 and CD8 T cell isolation Cells were blocked with 100 ml of Fc block (20 mg/ml; BD Bio- Splenocytes were washed once with autoMACS buffer (Miltenyi sciences) on ice for 15 min. Cells were then washed once. Sam- Biotec), counted, and suspended in 40 ml/107 cells autoMACS ples were incubated on ice for 30 min with 12.5 mg/ml 3G9/OVA buffer and 5 ml of CD8 or CD4 T cell biotin Ab mixture (Miltenyi and other appropriate Abs in 100 ml (see Supplemental Table I, Biotec)/107 cells. Cells were incubated for 10 min at 4°C, at which top) and mixed at 15 min into the incubation (14). Samples were Downloaded from point 30 mlofautoMACSbuffer/107 cells and 10 ml of anti-biotin washed twice. Biotinylated Abs were then detected with 2 mg/ml microbeads (Miltenyi Biotec)/107 cells were added. Cells were streptavidin/Brilliant Violet 510 (BD Biosciences), and 3G9/OVA incubated for 15 min at 4°C, washed with 10 ml of autoMACS was detectedwith 1:100donkeyanti-human IgG(H+L) F(ab9)2/PE buffer, and passed through an LS column (Miltenyi Biotec). T cells (Jackson ImmunoResearch). Samples were washed twice and were eluted from the column, suspended in RPMI-10, and incubated in 1 mg/ml DAPI for 15 min on ice. Samples were filtered http://www.immunohorizons.org/ counted. through a 35-mm cell strainer prior to data collection. Data were collected on a BD LSRII flow cytometer with five lasers (355, 405, T cell proliferation assay based on BrdU incorporation 488, 561, and 640 nm) and analyzed using FlowJo software CD11c+ DCs (or CD8a+ DCs, CD8a2 DCs, or bone marrow– (version 8.8.6). derived DCs [BMDCs], as indicated) were treated with the indicated Ags or Abs in a flat-bottom 96-well plate and incubated Analysis of maturation markers on APC populations at37°C.After2h,DCswerematuredwith0.5mg/ml LPS. The Splenocytes were added to a 96-well V-bottom plate, with 2 3 106 following day, T cells were added to the DCs. After an additional cells per well. The plate was centrifuged at 486 3 g for 5 min, and 72 h, BrdU labeling reagent (BD Biosciences) was added to each cells were washed one time in PBS (pH 7.2) containing 2% FBS. well at a concentration of 10 mM. The next day, a BrdU detection This buffer was used for all subsequent washing and dilution steps. by guest on September 27, 2021 ELISA (BD Biosciences) was performed. Absorbance of each well Cells were blocked with 100 mlofFcblock(20mg/ml; BD at 450 nm was detected using an EMax precision microplate Biosciences) on ice for 15 min. Cells were then washed once. reader (Molecular Devices), and the results were analyzed using Samples were incubated on ice for 30 min with appropriate Abs in GraphPad Prism 7 software. 100 ml (Supplemental Table I, top) and mixed at 15 min into the incubation (14). Samples were washed twice. The appropriate Ab Assessment of T1D (Supplemental Table I, middle) was added, and biotinylated Abs Female mice were monitored weekly from 4 to 30 wk for glucosuria were detected with 2 mg/ml streptavidin/Brilliant Violet 510 (BD using Diastix reagent strips (Bayer). Mice were considered dia- Biosciences). Samples were washed twice, incubated in 1 mg/ml betic following two consecutive positive tests. The first positive DAPI for 15 min on ice, and filtered through a 35-mm cell strainer test was recorded as the date of diabetes onset. Based on our prior prior to data collection. Data were collected on a BD LSRII flow studies with NOD mice, this corresponds to a blood glucose value cytometer with five lasers (355, 405, 488, 561, and 640 nm) and at onset of 467 6 121 mg/dl (n =11). analyzed using FlowJo software (version 8.8.6).

IFN-g ELISPOT assay Anti-insulin Western blot Islets were isolated from pancreata of NOD.hCD205 mice as Anti-mCD205 Abs were run on a 4–15% Tris–HCl gel (Bio-Rad) described (25) and cultured with IL-2 for 7 d. After 6 d, CD11c+ DCs and then either stained with Biosafe Coomassie (Bio-Rad) or were isolated from NOD mice, treated with peptides of interest, transferred to a PVDF membrane (Bio-Rad). The membrane was and maturedwith 0.5 mg/ml LPS. DCs were incubated overnight at blocked with 5% nonfat dry milk (Santa Cruz Biotechnology) in 37°C. An ELISPOT plate (EMD Millipore) was coated overnight 0.05% Tween 20/PBS. The membrane was then incubated with with anti–IFN-g (R4-6A2; BD Biosciences). The following day, guinea pig anti-insulin (Abcam) diluted 1:200 in blocking buffer. 2 3 104 islet-infiltrating cells were added to each well containing After a 1-h wash with 0.05% Tween 20/PBS, the membrane was treated DCs. The mixed cell population was added to the Ab- incubated with rabbit anti–guinea pig IgG–alkaline phosphatase coated ELISPOT plate and incubated at 37°C for 48 h. After this (Abcam) diluted 1:1000 in blocking buffer. The membrane was time, an IFN-g ELISPOT assay was performed as described (25). then washed for 40 min with 0.05% Tween 20/PBS and 20 min Spots were counted using an automated ELISPOT reader with PBS. Detection reagent was prepared by dissolving one tablet

https://doi.org/10.4049/immunohorizons.1900014 240 PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES ImmunoHorizons

of SigmaFast BCIP/NBT (Sigma-Aldrich) in 10 ml of H2Oand OT-I proliferation in response to SIINFEKL, the peptide recog- added to the membrane. After sufficient color development, the nized by OT-I, but could not induce proliferation in response to reaction was stopped by rinsing the membrane with H2O. Alter- 3G9/OVA (Fig. 1C), as expected. In contrast, C57BL/6.hCD205 natively, to verify the presence of the entire proinsulin protein, an DCs induced proliferation in response to both the peptide and Ab specific for the C-terminal region of the insulin A chain was 3G9/OVA (Fig. 1D). This indicated that the transgenic hCD205 used. The membrane was incubated with goat anti-insulin A (C-12; was functioning as expected. Furthermore, the proliferation Santa Cruz Biotechnology) diluted 1:500 in blocking buffer, seen was not due to an effect of 3G9 in the absence of OVA, followed by donkey anti-goat IgG–alkaline phosphatase (Santa as 3G9/SL9 induced no proliferation. We performed similar Cruz Biotechnology) diluted 1:5000 in blocking buffer. The experiments with OVA-specificCD4+ OT-II T cells, replacing membrane was then washed, and reactive bands were detected SIINFEKL with the peptide recognized by OT-II, ISQAVHAA as above. Membranes were imaged using an HP ScanJet 8200 HAEINEAGR. Once again, C57BL/6 DCs could only induce (Hewlett-Packard). OT-II T cell proliferation by presenting the OT-II peptide (Fig. 1E), whereas C57BL/6.hCD205 DCs induced proliferation IL-2 ELISA in response to 3G9/OVA, as well (Fig. 1F). Proliferation of OT-I Splenic CD11c+ DCs from NOD mice were plated at 2.5 3 104 cells (Fig. 1D) and OT-II cells (Fig. 1F) was observed even at the lowest Downloaded from per well in 100 ml of RPMI-10 in a 96-well flat-bottom plate and concentration of 3G9/OVA tested (0.1 mg/ml, or 0.4 nM), treated with 0.1 or 1 mMpeptidesInsB9–23 (SHLVEALYLVC suggesting that efficient presentation via hCD205 does not re- GERG; Mimotopes) or the BDC2.5 mimotope AHHPIWARMDA quire a large amount of Ag to be available. (26) (Mimotopes) or the indicated concentrations of Ag-linked To verify the in vitro function of hCD205 on the cDC1 and anti-mCD205 Abs. The cells were incubated for 2 h at 37°C, at cDC2 subsets individually, CD8a+ (Fig. 1G) and CD8a2 DCs http://www.immunohorizons.org/ which point the DCs were treated with 1 mg/ml LPS to induce (Fig. 1H) were isolated by magnetic separation from the spleens of maturation. Cells were incubated overnight at 37°C. The following C57BL/6.hCD205 mice and treated with 3G9/OVA or with the + day, the InsB9–23–specific cell line PCR1-10.18 (27) was added to indicated control reagents. OT-II CD4 T cells were added, and the DCs at 2.5 3 104 cells per well in 100 mlofRPMI-10. T cell proliferation was assessed by BrdU incorporation. We found Cocultures were incubated overnight at 37°C. IL-2 secretion that both of the cDC subsets could process hCD205-targeted Ags was measured by ELISA (eBioscience). Briefly, an ELISA plate and present them to T cells (Fig. 1G, 1H). was coated overnight with anti–IL-2 capture Ab. After blocking and washing, cell supernatants were added to the plate at 100 ml/ NOD mice with transgenic expression of hCD205 are well. Biotin–anti–IL-2 was used as a detection Ab, followed by susceptible to T1D incubation with avidin–HRP. 3,39,5,59-Tetramethylbenzidine To generate hCD205-transgenic NOD mice (NOD.hCD205), we by guest on September 27, 2021 substrate was added, and after color development, the reac- backcrossed the C57BL/6.hCD205 mice (18) with NOD mice for 12 tion was stopped by addition of 1 N H2SO4.ELISAplates generations. To verify that NOD.hCD205 mice remain susceptible were read at 450 nm, and IL-2 concentrations were calculated to T1D, we performed an incidence study, comparing female from an IL-2 standard curve. NOD.hCD205 mice to their nontransgenic NOD littermates (Fig. 2A). Mice were monitored weekly for glucosuria and were considered diabetic following two consecutive positive tests. The RESULTS two groups ofmice wereequallysusceptibleto T1D, indicating that the transgene does not interfere with the disease process. To hCD205-transgenic DCs process and present Ags delivered ensure that the disease remained of an autoimmune cause, we + + via hCD205 to both CD4 and CD8 T cells examined the specificities of T cells cultured from the islets of In hCD205-transgenic C57BL/6 mice, the transgene is under the NOD.hCD205 mice. NOD DCs were treated with the peptides control of a CD11c promoter, allowing expression on both the NRP-V7 and YQLENYCAL, which are mimotopes of b cell CD8a+ and CD8a2 classic myeloid DC subsets (18). As previously peptides recognized by 8.3- and AI4-like CD8+ T cells, respectively reported (18), the anti-hCD205 Ab 3G9 binds to CHO cells stably (19, 28). We found that two out of three mice contained 8.3-like expressing hCD205 (CHO/hCD205), with no binding to non- T cells, whereas one out of three contained AI4-like T cells, with transfected CHO cells or to CHO/mCD205 cells (Supplemental one mouse recognizing neither peptide (Fig. 2B). Individual NOD Fig. 1). To verify the functionality of the hCD205 transgene, we mice display a range of islet T cell specificities (29). Thus, it was developed anti-hCD205 linked to OVA (3G9/OVA). 3G9/OVA was both unsurprising and reassuring to see that range reflected in the produced in 293T cells (Fig. 1A), and its binding to hCD205 but not NOD.hCD205 mice, as well. mCD205 was verified (Fig. 1B). CD11c+ DCs were isolated from C57BL/6 or C57BL/6.hCD205 mice and treated with 3G9/OVA or APCs from NOD.hCD205 mice develop as is typical for 3G9 linked to the HIV-derived peptide SLYNTVATL (3G9/SL9) NOD mice followed by LPS. To determine whether Ag could be internalized, The next step was to ensure that incorporation of the hCD205 processed, and presented, we added OVA-specificCD8+ OT-I transgene had not impacted APC development. Relative amounts T cells to these cultures. We found that C57BL/6 DCs could induce of APCs and their maturation status are known to be important in

https://doi.org/10.4049/immunohorizons.1900014 ImmunoHorizons PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES 241 Downloaded from http://www.immunohorizons.org/ by guest on September 27, 2021

FIGURE 1. hCD205-transgenic DCs treated with 3G9/OVA can process and present Ag to both CD8+ and CD4+ T cells. (A) 3G9/OVA was produced in 293T cells by transient transfection, purified by protein G affinity chromatography, and characterized by SDS-PAGE (lane 1, m.w. standards; lane 2, 3G9/OVA). The H and L chains of 3G9/OVA are indicated. (B) Binding of 3G9/OVA to hCD205 was verified by flow cytometric staining of the indicated cell lines. (C–F) To assess the in vitro function of hCD205, CD11c+ DCs from either C57BL/6 (C and E) or C57BL/ 6.hCD205 mice (D and F) were treated with 3G9/OVA or with the indicated control reagents in a 96-well plate. OT-I CD8+ (C and D) (Continued)

https://doi.org/10.4049/immunohorizons.1900014 242 PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES ImmunoHorizons

FIGURE 2. NOD.hCD205 mice are susceptible to T1D. (A) Diabetes incidence curves for female NOD and NOD.hCD205 littermates are shown. p = 0.72 (Mantel–Cox). (B) To verify the presence of diabetogenic T cells in NOD.hCD205 mice, islet-infiltrating T cells from three female NOD.hCD205 mice were added to NOD DCs that were Downloaded from incubated with the indicated peptides, and T cell reactivity was measured by IFN-g ELISPOT. Graph displays stimulation index for each peptide (mean + SEM of triplicates); dotted line indicates a stimulation index of 2. TUM (KYQAVTTTL) is an irrelevant H2-Kd–binding peptide; TRL9 (TSPRNSTVL) is an irrelevant H2-Db–binding peptide.

T1D pathogenesis (13, 30), so we examined the status of several NOD mice expressed approximately two times the amount found http://www.immunohorizons.org/ majorAPCsubsetsbyflow cytometry: monocytes, monocyte- in the C57BL/6 background (Fig. 3C). Also, C57BL/6 mice had an derived DCs (MoDCs), plasmacytoid DCs (pDCs), and CD8a+ and mCD2052 population among their CD8a+ DCs, whereas all NOD CD8a2 DCs (Fig. 3A). Following a previously described strategy CD8a+ DCs expressed mCD205, suggesting that mice of different (14), we first gated on splenocytes negative for CD3e,CD19, backgrounds may regulate mCD205 differently. NKp46, and Ly6G to exclude T cells, B cells, NK cells, and We used the gating strategy illustrated in Fig. 3A to determine neutrophils, respectively. To examine the pDCs, we gated on whether the hCD205 transgene had an impact on any APC CD11c+ Siglec-H+ cells. Next, Siglec-H2 cells were defined based populations, either in the C57BL/6 or the NOD strains, in terms of on whether they were CD11chi or CD11bhi.CD11bhi cells were the percentage of splenocytes or the absolute number of cells further distinguished based on Ly6C or MHC II expression; (Fig. 4). No significant differences were found in either percent- CD11bhi Ly6C+ MHC II2 cells were considered monocytes, ages or absolute numbers of cells when comparing the transgenic by guest on September 27, 2021 whereas CD11bhi Ly6C2 MHC II+ cells were considered MoDCs. and parent strains. Thus, the hCD205 transgene had no impact on Meanwhile, CD11chi cells were gated on high MHC II expression, APC development. These experiments did reveal some differences and these cells were then separated based on expression of DCIR2 between the C57BL/6 and NOD backgrounds, consistent with or CD8a.CD11chi MHC IIhi CD8a2 DCIR2+ cells were considered previously reported findings (14). We found that there was a signif- CD8a2 DCs, whereas CD11chi MHC IIhi CD8a+ DCIR22 cells icant increase in both percentage and absolute number of pDCs were considered CD8a+ DCs. To confirm the identity of each in the NOD background (Fig. 4). However, despite previous subset, we examined a number of other markers for each (Fig. 3B). reports (14), we did not find significant increases in the percent- Forexample,pDCsareknowntoexpressCD4andMHCII(14), ages and absolute numbers of NOD MoDCs or decreases in CD8a+ which we observe, as well. Additionally, although MoDCs and DCs, as compared with C57BL/6 mice (Fig. 4). CD8a2 DCs both express CD11c, CD11b, and MHC II, they can be differentiated from each other by the DCIR2 and CD4 expression APCs from NOD.hCD205 mice have typical maturation of CD8a2 DCs, which MoDCs lack (14). As expected, only CD8a+ statuses for NOD mice DCs exhibited significant mCD205 expression (14). Interestingly, Although the percentages and absolute numbers of the five APC although the expression of mCD205 in CD8a+ DCs from trans- subsets do not differ between transgenic mice and their parent genic mice did not differ from their nontransgenic littermates, strains (Fig. 4), it is possible that these strains differ functionally.

or OT-II CD4+ T cells (E and F) were added (10,000 cells per well), and T cell proliferation was assessed by BrdU incorporation, which was measured by ELISA. Graphs display mean absorbance 6 SEM at the indicated DC:T cell ratios; each point represents a mean of three replicates. (C) C57BL/6 DCs and OT-I CD8+ T cells. (D) C57BL/6.hCD205 DCs and OT-I CD8+ T cells. (E) C57BL/6 DCs and OT-II CD4+ T cells. (F) C57BL/6.hCD205 DCs and OT-II CD4+ T cells. (G and H) To assess the in vitro function of hCD205 on the cDC1 and cDC2 subsets individually, CD8a+ (G) and CD8a2 (H) DCs were isolated by magnetic separation from the spleens of C57BL/6.hCD205 mice and treated with 3G9/OVA or with the indicated control reagents. OT-II CD4+ T cells were added (10,000 cells per well), and T cell proliferation was assessed by BrdU incorporation. The DC:T cell ratio was 2:1. Graphs display mean 6 SEM.

https://doi.org/10.4049/immunohorizons.1900014 ImmunoHorizons PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES 243 Downloaded from http://www.immunohorizons.org/ by guest on September 27, 2021

FIGURE 3. The presence of hCD205 does not alter APC phenotype. Splenocytes from C57BL/6 and NOD mice with and without hCD205 (three female mice per group; 15–19 wk old) were analyzed by flow cytometry for expression of APC phenotypic markers including CD11c, CD11b, MHC II, Ly6C, CD8a, and DCIR2. (A) Gating strategy used to identify pDCs, MoDCs, monocytes (Mono), and CD8a+ and CD8a2 DCs. (B) Representative histograms for expression of (from left to right) mCD205, CD4, CD11b, CD11c, H2-Ag7, and H2-Ab by (top to bottom) CD8a+ DCs, CD8a2 DCs, pDCs, MoDCs, and Mono. (C) Relative mCD205 expression on CD8a+ DCs from each mouse strain compared with C57BL/6 mice. Graph depicts mean + SEM; p values are indicated (t test).

https://doi.org/10.4049/immunohorizons.1900014 244 PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES ImmunoHorizons

status in this study does not support this definition (13, 36, 37). Monocytes from NOD and C57BL/6 mice have equivalent expression of CD80, CD86, CD40, PD-L1, and class I MHC; the same is true for pDCs (Fig. 5B). In sum, NOD CD8a+ DCs, MoDCs, pDCs, and monocytes have a similar maturation status as their C57BL/6 counterparts, whereas CD8a2 DCs from NOD mice appear to be more mature. However, it should be noted that each subset was examined at steady state without addition of exogenous maturation stimuli, so although these DCs may have increased expression of maturation markers, they are likely still immature or at least not fully differentiated. Finally, in addition to finding minimal differences between NOD and C57BL/6 APCs, there are no differences when comparing transgenic and nontransgenic mice. We therefore find that APCs from NOD.hCD205 mice develop as is typical for Downloaded from NOD APCs.

DCs from NOD.hCD205 mice process and present Ags As the hCD205 transgene had no detectable impact on APC development or maturation in NOD mice, we next tested the http://www.immunohorizons.org/ function of NOD.hCD205 CD11c+ DCs. DCs were treated with either NRP-V7 or YQLENYC, peptides recognized, respectively, FIGURE 4. APCs from NOD.hCD205 mice develop similarly to APCs by the 8.3 and AI4 CD8+ T cell clones (19, 28), and matured with from NOD mice. LPS. CD8+ T cells from either 8.3 or AI4 TCR-transgenic mice Splenocytes from the indicated strains of mice (three female mice per were added to these DCs, and their proliferation was assessed group; 15–19 wk old) were analyzed by flow cytometry, and APC subsets (Fig. 6A, 6B). We found that NOD.hCD205 DCs were able to in- were identified following the gating strategy shown in Fig. 3A. (A)Per- duce proliferation of these cells to the same extent as NOD DCs, centages of splenocytes and (B) numbers of cells among splenocytes are indicating that they remain sensitive to maturation stimuli and can shown. Graphs depict mean + SEM; p values are indicated (t test). elicit a T cell response. Free peptides most likely displace peptides already present on surface MHC rather than being taken up and by guest on September 27, 2021 Knowing that DC maturation can be important to T1D pathogen- loaded onto new peptide–MHC complexes. We therefore next esis (13, 30), we examined the maturation status of each subset by examined the ability of NOD.hCD205 DCs to process and present measuring steady-state expression of several maturation markers, Ag taken up by endocytosis by using anti-mCD205 that was linked including CD80, CD86, CD40, PD-L1, and class I MHC and MHC to NRP-V7 (Fig. 6C) (8). We found that this, too, led to the II (31, 32). Expression for each strain was compared with C57BL/6 proliferation of 8.3 CD8+ T cells, indicating that endocytosed Ag mice. Once again, the presence of the transgene did not impact the can be taken up, processed, and cross-presented. expression of any marker tested (Fig. 5). There were, however, a number of differences when comparing mice of the NOD and NOD.hCD205 mice show reduced expression of hCD205 C57BL/6 backgrounds, although in general the differences were compared with C57BL/6.hCD205 mice slight, as has been previously reported (33–35). CD8a+ DCs do not The hCD205 transgene is under the control of a synthetic CD11c differ in expression of any of the aforementioned markers on a promoter containing both C57BL/6 and 129 sequences (18). CD8a+ comparison of the two strains, suggesting that they are of similar and CD8a2 DCs have high CD11c expression, as shown in the maturationstatus(Fig.5B).Meanwhile,CD8a2 DCs from NOD above gating strategy (Fig. 3A). However, MoDCs and pDCs have mice display significantly higher PD-L1 and class I MHC expression intermediate levels of CD11c expression, as well. Thus, hCD205 than that seen in the C57BL/6 background (Fig. 5B). It would may be expressed not only by the former subsets but by the latter therefore appear that CD8a2 DCs from NOD mice are more mature two also. We therefore used 3G9/OVA to probe for expression than those of C57BL/6 mice. Given their expression of PD-L1, they of hCD205 (Fig. 7A). We found that monocytes are not stained may be particularly useful in the generation of T cell tolerance. with 3G9, an unsurprising result, as they do not express CD11c. Similar to the CD8a+ DC subset, NOD MoDCs do not signifi- Similarly, pDCs, although they do express CD11c, did not have cantly differ in their expression of any examined maturation marker detectable hCD205 expression, consistent with what has been when compared with C57BL/6 mice (Fig. 5B). Thus, despite a trend previously found in C57BL/6.hCD205 mice (18). In contrast, toward somewhat higher numbers of this subset in the NOD MoDCs exhibit high expression of hCD205. As expected, both background (Fig. 4B), these cells have the same maturation status as CD8a+ and CD8a2 DCs express hCD205; however, hCD205 those found in C57BL/6 mice. This is ofinterest,asthissubsetisoften expression in NOD.hCD205 mice is only ;25% of that observed in defined as inflammatory DCs, whereas we find that their maturation the C57BL/6.hCD205 strain (Fig. 7B). Finally, whereas almost all

https://doi.org/10.4049/immunohorizons.1900014 ImmunoHorizons PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES 245 Downloaded from http://www.immunohorizons.org/ by guest on September 27, 2021

FIGURE 5. APCs from NOD.hCD205 mice have a similar maturation status as NOD mice. Expression of maturation markers by splenocytes from C57BL/6 and NOD mice with and without hCD205 (three female mice per group; 15–19 wk old) was investigated by flow cytometry. (A) Representative histograms for expression of (from left to right) CD80, CD86, CD40, PD-L1, and H2-Db by (top to bottom) CD8a+ DCs, CD8a2 DCs, pDCs, MoDCs, and monocytes (Mono). (B) Relative expression of the indicated markers by each cell type in the different mouse strains. Expression for each subset was compared with the C57BL/6 strain. Graphs depict mean + SEM; p values are indicated (t test).

https://doi.org/10.4049/immunohorizons.1900014 246 PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES ImmunoHorizons

regardless of their activation status (based on CD62L and CD44 expression) or the presence of CD11c (Supplemental Fig. 2C, 2D). Thus, as previously suggested for the C57BL/6.hCD205 mice (18), expression of hCD205 is limited to DCs in NOD.hCD205 mice.

DCs from NOD.hCD205 mice process and present Ag delivered via hCD205 to T1D-relevant T cells We had thus far established that NOD.hCD205 mice behaved as typical NOD mice in their T1D susceptibility, APC development and maturation, and Ag-presenting abilities. The final step was to ensure that hCD205 functioned in these mice as already shown in the C57BL/6 background (Fig. 1). This was of particular importance, given our finding that hCD205 expression in the NOD.hCD205 strain was reduced relative to that observed in C57BL/6.hCD205 mice(Fig.7B).Fortheseexperiments,3G9waslinkedtoYQLENYCAL Downloaded from (3G9/AL), a mimotope peptide recognized by the islet-reactive CD8+ T cell clone AI4 (19) (Fig. 8A). CD11c+ splenic DCs from NOD and NOD.hCD205 mice were treated with 3G9/AL or 3G9/OVA followed by LPS. CD8+ AI4 T cells were then added to the cultures, and T cell proliferation was measured by BrdU incorporation http://www.immunohorizons.org/ (Fig. 8B). Robust proliferation was observed in the presence of splenic DCs isolated from NOD.hCD205 mice and treated with 3G9/AL. Similar findings were observed when BMDCs were used (Fig. 8C). Thus, despite lower transgene expression in the NOD background, hCD205 expressed by NOD mice is functional. The NOD.hCD205 strain can thus serve as a preclinical model for the evaluation of hCD205-mediated Ag delivery therapeutics.

Development of reagents to modulate the insulin-specific autoimmune response in T1D by guest on September 27, 2021 Because proinsulin is a critical b cell Ag in T1D (42–45), we FIGURE 6. CD11c+ DCs from NOD.hCD205 mice are able to process developed and characterized proinsulin-linked anti-mCD205 in a and present Ag to diabetogenic CD8+ T cells. proof-of-principle study to determine whether Ab-linked pro- (A–C) To assess the function of DCs from NOD.hCD205 mice, NOD or insulin could be processed and presented by DCs. Proinsulin 2 NOD.hCD205 CD11c+ DCs were treated with 1 mM NRP-V7, 1 mM (rather than proinsulin 1) was used for these studies because of its YQLENYCAL (AL), 1 mg/ml anti–mCD205/NRP-V7, or appropriate con- closer similarity to hIns. To produce anti-mCD205/Ins2, we trols in a 96-well plate. Either 8.3 (A and C) or AI4 (B) CD8+ T cells were cloned the Ins2 cDNA in frame with the C terminus of the H chain added, and T cell proliferation was assessed by BrdU incorporation, of an anti-mCD205 Ab (derived from NLDC-145) in which the which was quantified by ELISA. Graphs display mean absorbance 6 SEM mouse IgG1 C region had been mutated to interfere with Fc fl at the indicated DC:T cell ratios; each point indicates a mean of three receptor binding (9). The exible linker (GGGGS)3 (20) was replicates. (A) NOD or NOD.hCD205 DCs and 10,000 8.3 CD8+ Tcells inserted between the H chain and proinsulin to facilitate proinsulin per well. (B) NOD or NOD.hCD205 DCs and 10,000 AI4 CD8+ T cells per folding, which we determined was necessary for the expression of well. (C) NOD.hCD205 DCs and 20,000 8.3 CD8+ T cells per well. theAbwhenproducedin293Tcells.Anti-mCD205/Ins2was characterized by SDS-PAGE and showed an increase in the m.w. of the H chain compared to that of bovine g-globulin due to the C57BL/6.hCD205 MoDCs are positive for hCD205, only ;75% of attached proinsulin (Fig. 9A). Western blotting with a polyclonal Ab NOD.hCD205 MoDCs express the transgene (Fig. 7A). to insulin revealed binding to the H chain of anti-mCD205/Ins2 but Immune cells other than DCs can also express CD11c, including not anti-mCD205/OVA (Fig. 9B). Similar results were obtained some activated T cells (38–41).Forthisreason,weexamined using an Ab against the C terminus of the insulin A chain (Fig. 9B). CD11c+ splenocytes also positive for CD3e, CD19, NKp46, or Ly6G Anti-mCD205/Ins2 bound similarly to CHO/mCD205 cells, as did (designated Lineage+ CD11c+ cells) for expression of the hCD205 the well-characterized anti-mCD205/OVA (15) (Fig. 9C, left). The transgene in our newly developed NOD.hCD205 mice. No evidence presence of proinsulin on the bound Ab was verified using a for expression of the transgene was observed (Supplemental Fig. secondary Ab specific for proinsulin (Fig. 9C, right). + + g7 + 2A, 2B). When CD8 and CD4 T cells were specifically examined, InsB9–23 presented by H2-A is a major target of CD4 Tcells we did not observe any population of cells expressing hCD205, in NOD mice, essential for diabetes development (45, 46), and also

https://doi.org/10.4049/immunohorizons.1900014 ImmunoHorizons PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES 247

atargetofCD4+ T cells in humans when presented by the structurally similar HLA-DQ8 (47–49). We evaluated processing

and presentation of InsB9–23 by NOD mouse DCs from anti- mCD205/Ins2 using the cognate T cell hybridoma PCR1-10.18 (27). PCR1-10.18 cells released IL-2 in response to anti-mCD205/ Ins2–treated DCs (Fig. 9D), demonstrating that Ab-linked pro- insulin epitopes are functionally processed and presented to T cells. Having found that proinsulin-linked anti-mCD205 could be produced in vitro and that proinsulin-derived T cell epitopes could be processed and presented when DCs were treated with anti- mCD205/Ins2 (Fig. 9D), the same strategy was used to link Ins2 to the C terminus of the H chain of the human anti-hCD205 Ab 3G9 (18). The resulting anti-hCD205/Ins2 was characterized by SDS- PAGE (Fig. 9E, left) and was found to bind similarly to CHO cells Downloaded from modified to express hCD205, as did anti-hCD205/OVA (Fig. 9E, right). hIns was similarly linked to the C terminus of the H chain of anti-hCD205, and anti-hCD205/hIns was successfully produced in 293T cells (Fig. 9F, left). We verified that, as expected, the presence of hIns did not disrupt the binding of the Ab to hCD205 http://www.immunohorizons.org/ (Fig. 9F, right). The availability of this reagent elevates the potential translational impact of this Ag delivery strategy.

DISCUSSION

Autoreactive T cells are responsible for the destruction of the insulin-producing b cells, leading to the development of T1D (1). One potential strategy for preventing disease is to inhibit the activation of, and/or induce tolerance in, these autoreactive T cells (5, 6). As both CD4+ and CD8+ T cells are required for T1D to occur by guest on September 27, 2021 (1, 50), it will likely be necessary to inhibit the function of both of these T cell subsets. Under steady-state conditions, DCs can present Ag to both CD4+ and CD8+ T cells in a tolerogenic manner, resulting in T cell deletion, anergy, and/or the induction and expansion of regulatory T cells (5, 6). This, therefore, represents an attractive method for preventing the pathogenicity of autoreactive T cells in T1D. Previous work in the NOD mouse has shown that Ags can be delivered to the CD8a+ or CD8a2 DC subsets by linking an Ag to anti-mCD205 (8–10, 51) or anti-DCIR2 (11), respectively. In these cases, CD8a+ DCs induced tolerance in CD8+ T cells (8, 9), and CD8a2 DCsdidthesameforCD4+ T cells (11). The ability of CD8a+ DCs to induce CD4+ T cell tolerance in this strain is less clear, as there are conflicting reports in the literature on this point (10, 51). Other studies, using Ag delivery without DC targeting, were also successful in impacting the disease process in NOD mice; however, such studies have failed to effectively translate to humans (4). By improving the approach, both by delivering Ag directly to DCs and by using a more clinically relevant model system, we can

FIGURE 7. CD8a+, CD8a2, and MoDCs from NOD.hCD205 mice express hCD205. 2 (A) Expression of hCD205 by splenocytes from C57BL/6 and NOD mice CD8a+, CD8a , and MoDCs from C57BL6 (white) and NOD (black) with and without hCD205 (three female mice per group; 15–19 wk old) hCD205-transgenic mice. Graph depicts mean MFI + SEM; p values are was analyzed by flow cytometry. (B) hCD205 expression by splenic indicated (t test). Mono, monocytes.

https://doi.org/10.4049/immunohorizons.1900014 248 PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES ImmunoHorizons Downloaded from

FIGURE 8. CD11c+ DCs from NOD.hCD205 mice are able to process and present Ag delivered via hCD205. (A) 3G9/AL was produced in 293T cells by transient transfection, purified by protein G affinity chromatography, and characterized by SDS-PAGE http://www.immunohorizons.org/ (lane 1, m.w. standards; lane 2, 3G9/AL; lane 3, bovine g-globulin). The H and L chains of 3G9/AL are indicated. (B) Splenic CD11c+ DCs (50,000 per well) or (C) BMDCs (25,000 per well) from the indicated mice were treated with 3G9/OVA or 3G9/AL [0.2 mg/ml in (B) or as indicated in (C)] in a 96-well plate. AI4 CD8+ T cells were added at a DC:T cell ratio of 1:1, and T cell proliferation was assessed by BrdU incorporation, which was quantified by ELISA. Graphs depict mean + SEM of technical triplicates; p values are indicated (t test). improve the likelihood that the treatment will translate from mice ideal endocytic receptor for the targeting of both DC subsets. to humans. Furthermore, using one reagent to target multiple subsets is Tothat end, we have developed an NOD-based mouse model an improvement over needing to use a different reagent for for T1D having transgenic expression of hCD205. Aside from each subset. In humans, BDCA-3+ DCs express ;2-fold higher + the very fact that we focus on hCD205 rather than on murine CD205 on their surface than do BDCA-1 DCs (52). Inter- by guest on September 27, 2021 endocytic receptors, there are several reasons to expect this estingly, in the hCD205-transgenic C57BL/6 and NOD mice, model to have a higher translational potential than the DC- the CD8a+ DC subset trended toward enhanced surface targeted treatments mentioned above. Whereas in mice, CD205 expression of hCD205 compared with the CD8a2 subset is expressed only by the CD8a+ subset, hCD205 is expressed by (Fig. 7B; p = 0.206 and 0.131 for the C57BL/6 and NOD strains, both the BDCA-1+ and the BDCA-3+ DCsubsetsinhumans(13, respectively). 52). Although less studied than the murine DC subsets, BDCA- In developing the NOD.hCD205 model, we examined whether 1+ DCs are thought to parallel the function of the murine the transgene had any impact on T1D or on the status of APCs. We CD8a2 subset, whereas BDCA-3+ DCs may serve the same found that NOD.hCD205 mice had a T1D incidence indistinguish- function as the murine CD8a+ subset (13). The two DC subsets able from that of NOD littermates and contained AI4-like and are believed to be specialized for presentation to either CD4+ 8.3-like T cells in their islet infiltrate. We further found that or CD8+ T cells, respectively, although neither DC subset is the presence of the transgene had no impact on the percentages limited to presentation to one type of T cell (15). CD205 can or absolute numbers of any examined APC subset, including serve an important practical role in this context, as Ag monocytes, MoDCs, pDCs, and CD8a+ and CD8a2 DCs, nor was endocytosed by human DCs via CD205 can be processed and there any impact on maturation status. In all cases, APCs from presented on MHC II, as is typical for extracellular Ags; CD205 NOD.hCD205 mice were indistinguishable from those from NOD is also able to mediate cross-presentation, with the result that mice; the same can be said when comparing C57BL/6.hCD205 peptidescanbepresentedonclassIMHC,aswell(52,53).Asa mice to their nontransgenic counterparts. As expected, the result, Ag delivered by anti-CD205 can impact both CD4+ and amounts of several subsets differed when comparing the C57BL/ CD8+ T cells, both of which will likely need to be targeted for 6 and NOD backgrounds (14). effective T1D therapy (1, 50). A drawback of using mCD205 Therewas,however,onesurprisingresult.Wefoundthat,in targeting as a model system is that CD8a+ DCs may not be able the NOD background, CD8a+ and CD8a2 DCs had reduced to present to CD4+ Tcellseffectively, whereas the CD8a2 surface hCD205 expression compared with transgenic C57BL/ subset lacks mCD205 (13, 15). This divide is not an issue in 6 mice. This was despite a trend toward increased expression of the case of human DCs, however, and CD205, therefore, is an endogenous CD11c in the NOD strain (Supplemental Fig. 3A).

https://doi.org/10.4049/immunohorizons.1900014 ImmunoHorizons PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES 249 Downloaded from http://www.immunohorizons.org/ by guest on September 27, 2021

FIGURE 9. Proinsulin delivered to DCs via CD205 can be processed and presented to T cells. (A) Anti-mCD205/Ins2 was produced in 293T cells by transient transfection and purified from the culture supernatant. SDS-PAGE analysis was performed under reducing conditions (lane 1, m.w. standards; lane 2, bovine g-globulin; lane 3, anti-mCD205/Ins2; lane 4, anti-mCD205/OVA). The H and L chains of anti-mCD205/Ins2 are indicated. (B) Western blot analysis using a polyclonal guinea pig anti-insulin Ab (lanes 1–3) or a polyclonal goat anti-insulin A chain Ab (lanes 4–6). Lanes 1 and 4, anti-mCD205/OVA; lanes 2 and 5, anti-mCD205/Ins2; lanes 3 and 6, bovine g-globulin. (C) CHO/mCD205 cells were stained with the indicated Abs, followed by a labeled anti-IgG (left) or an anti-proinsulin (right) secondary Ab. (D)Splenic DCs from NOD mice were treated with the indicated peptides or Abs and then cocultured with PCR1-10.18 cells for 24 h. T cell response was quantified by IL-2 ELISA of culture supernatants. Graph depicts mean + SD of two independent experiments; p values are indicated (t test). (E)3G9/ Ins2 was produced in 293T cells by transient transfection, purified using protein G, and analyzed by SDS-PAGE. Left, lane 1, m.w. standards; lane 2, 3G9/ Ins2; lane 3, bovine g-globulin. The H and L chains of 3G9/Ins2 are indicated. Right, CHO/hCD205 cells were stained with (Continued)

https://doi.org/10.4049/immunohorizons.1900014 250 PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES ImmunoHorizons

The hCD205 transgene is under the control of a synthetic DC-targeted therapies that can now be developed using the CD11c promoter (GenBank accession number: DQ658851) NOD.hCD205 mice. containing both C57BL/6 and 129 sequences (18). When we WehaveshownthatIns2andhInscanbelinkedtoCD205- compared the synthetic CD11c promoter sequence with the specific Abs and readily purified from 293T cell supernantants. corresponding regions of the C57BL/6 and NOD genomes using 3G9/Ins2 will be of immediate use in investigations in which we Ensembl (54), only limited variation was observed for either will examine the impact of treatment with this Ab on T1D strain, suggesting that the reduction in hCD205 expression in incidence, T cell tolerance, and/or deletion of insulin-specific the NOD strain is unlikely to be due to reduced promoter T cells. Of particular interest will be tracking the presence of activity. Additionally, to be sure that the hCD205 transgene had particular T cell clones. The latter will be possible through the not undergone some alteration in the NOD mice that led to use of peptide–MHC tetramers; the epitopes of a number of reduced surface expression of the protein, we crossed hemi- insulin-specific T cell clones have been identified, including the zygous transgene-positive C57BL/6 mice with transgene- CD8+ T cell clones AI4 and G9C8 (19, 64). Also of interest is the negative NOD mice. Similar to hemizygous NOD mice, the impact the prevention of anti-insulin reactivity might have on transgene-positive F1 progeny showed a reduced level of non–insulin-specific T cells. If insulin is important as an surface hCD205 expression when compared with hemizygous initiating Ag and anti-insulin attack is prevented, we may also Downloaded from C57BL/6 mice (Supplemental Fig. 3B). These results suggest see a decrease in the population of T cells specific for other b that surface expression of hCD205 is under the control of one cell Ags. Disease prevention and reversal studies with 3G9/Ins2 or more genes that differ between C57BL/6 and NOD mice and will require consideration and optimization of multiple that the transgene in our NOD colony has not undergone parameters, including dose, timing, and frequency of admin- an untoward alteration leading to reduced transcription. istration. Finally, we have developed anti-hCD205 linked to http://www.immunohorizons.org/ This finding could be related to the observations that APC hIns. By crossing our NOD.hCD205 mice with mice transgeni- populations from autoimmune-prone mice and humans can cally expressing human insulin (65) and treating them with show alterations in phenotype and/or function when compared 3G9/hIns, we can further improve the translational impact of with control individuals (30). In any event, the critical point is our model. that despite a decrease in expression, hCD205 still allows for In sum, we have generated and characterized a mouse model of processing and presentation of Ag delivered via 3G9 in the T1D that incorporates the transgenic expression of hCD205. These transgenic NOD strain, validating its potential as a preclinical mice represent a unique, well-characterized, anduseful tool for the model. preclinical development and optimization of DC-directed T1D We envision a number of future uses for the NOD.hCD205 therapies. Of note, 3G9 linked to the tumor Ag NY-ESO-1 was mousemodel. Previous DC-targetedtreatmentstudieshave largely recently tested in humans for its ability to elicit an immune by guest on September 27, 2021 focused on the delivery of b cellmimotopestoeitherCD8a+ or response when administered along with an adjuvant to induce DC CD8a2 DCs to specifically target CD8+ or CD4+ Tcells(8,9,11).In activation, and it was found to be effective and safe (66). This our model, in which both subsets express hCD205, it would be strategy was first tested for safety and efficacy using C57BL/ ideal to delivera hCD205-linked Ag that includes epitopesrelevant 6.hCD205 as the preclinical model (18). Related approaches to to both CD4+ and CD8+ T cells. Proinsulin is the most appropriate combat T1D will use Ag-linked Abs without such an adjuvant Ag choice, as establishment of tolerance to insulin can lead to because the desired outcome will be tolerance. Nonetheless, this prevention of T1D (42, 43), and remission of established disease precedent for the use of Ag-linked anti-CD205 in humans, coupled (55), in NOD mice. Indeed, studies in mice suggest that it may be with the availability of our well-characterized NOD.hCD205 the long sought-after initiating Ag in T1D (42–45). Furthermore, in mouse strain, should facilitate the translation of 3G9/hIns and humans, the non-MHC locus that confers the strongest suscep- related reagents to clinical practice. tibility to T1D is the insulin gene regulatory region (56), and disease-associated alleles are correlated with reduced thymic expression (57, 58). Finally, CD4+ and CD8+ T cell responses to DISCLOSURES insulin are observed in T1D patients (59–62), in whom the reduced thymic insulin expression correlates with impaired negative L.-Z.H. and T.K. are employees and shareholders of Celldex selection of high-avidity insulin-specificTcells(63).Inducinga Therapeutics. The other authors have no financial conflicts of tolerogenic response to insulin is, therefore, a primary goal of the interest.

3G9/Ins2 or 3G9/OVA followed by a labeled anti-human IgG secondary Ab. Binding to hCD205 was assessed by flow cytometry. (F) 3G9/hIns was produced in 293T cells, and the purified Ab was analyzed by SDS-PAGE. Left, lane 1, m.w. standards; lane 2, 3G9/hIns; lane 3, bovine g-globulin. The H and L chains of 3G9/hIns are indicated. Right, 3G9/hIns was added to CHO/hCD205 cells, and binding was assessed by flow cytometry using a labeled anti-human IgG secondary Ab.

https://doi.org/10.4049/immunohorizons.1900014 ImmunoHorizons PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES 251

ACKNOWLEDGMENTS 16. Collin, M., and V. Bigley. 2018. Human dendritic cell subsets: an up- date. Immunology 154: 3–20. We thank Ralph Steinman (The Rockefeller University) for providing the 17. Kato, M., K. J. McDonald, S. Khan, I. L. Ross, S. Vuckovic, K. Chen, C57BL/6.hCD205 mice and the plasmids encoding the anti-mCD205 H and D. Munster, K. P. MacDonald, and D. N. Hart. 2006. Expression of L chains, Chae Gyu Park (Yonsei University College of Medicine) for human DEC-205 (CD205) multilectin receptor on leukocytes. Int. providing the CHO/mCD205 and CHO/hCD205 cell lines, Fernando Immunol. 18: 857–869. Macian (Albert Einstein College of Medicine) for providing the Rag1- 18. Cheong, C., J. H. Choi, L. Vitale, L. Z. He, C. Trumpfheller, deficient OT-I TCR-transgenic C57BL/6 mice, and John Kappler (National L.Bozzacco,Y.Do,G.Nchinda,S.H.Park,D.B.Dandamudi,etal. Jewish Health) for providing the PCR1-10.18 cell line. 2010. Improved cellular and humoral immune responses in vivo following targeting of HIV Gag to dendritic cells within human anti-human DEC205 monoclonal antibody. Blood 116: 3828–3838. REFERENCES 19. Lamont, D., G. Mukherjee, P. R. Kumar, D. Samanta, C. G. McPhee, T. W. Kay, S. C. Almo, T. P. DiLorenzo, and D. V. Serreze. 2014. 1. Pugliese, A. 2017. Autoreactive T cells in type 1 diabetes. J. Clin. Compensatory mechanisms allow undersized anchor-deficient class I Invest. 127: 2881–2891. MHC ligands to mediate pathogenic autoreactive T cell responses. 2. Mathieu, C., P. Gillard, and K. Benhalima. 2017. Insulin analogues J. Immunol. 193: 2135–2146.

in type 1 diabetes mellitus: getting better all the time. Nat. Rev. 20. Trinh, R., B. Gurbaxani, S. L. Morrison, and M. Seyfzadeh. 2004. Downloaded from Endocrinol. 13: 385–399. Optimization of codon pair use within the (GGGGS)3 linker sequence 3. Tooley, J. E., F. Waldron-Lynch, and K. C. Herold. 2012. New and results in enhanced protein expression. Mol. Immunol. 40: 717–722. future immunomodulatory therapy in type 1 diabetes. Trends Mol. 21. Hawiger, D., K. Inaba, Y. Dorsett, M. Guo, K. Mahnke, M. Rivera, Med. 18: 173–181. J. V. Ravetch, R. M. Steinman, and M. C. Nussenzweig. 2001. Dendritic 4. Babad, J., A. Geliebter, and T. P. DiLorenzo. 2010. T-cell autoantigens cells induce peripheral T cell unresponsiveness under steady state in the non-obese diabetic mouse model of autoimmune diabetes. conditions in vivo. J. Exp. Med. 194: 769–779. http://www.immunohorizons.org/ Immunology 131: 459–465. 22. Serreze, D. V., H. D. Chapman, D. S. Varnum, M. S. Hanson, 5. Audiger, C., M. J. Rahman, T. J. Yun, K. V. Tarbell, and S. Lesage. P.C.Reifsnyder,S.D.Richard,S.A.Fleming,E.H.Leiter,andL.D.Shultz. 2017. The importance of dendritic cells in maintaining immune tol- 1996. B lymphocytes are essential for the initiation of T cell-mediated erance. J. Immunol. 198: 2223–2231. autoimmune diabetes: analysis of a new “speed congenic” stock of 6. Iberg, C. A., A. Jones, and D. Hawiger. 2017. Dendritic cells as in- NOD.Ig m null mice. J. Exp. Med. 184: 2049–2053. – ducers of peripheral tolerance. Trends Immunol. 38: 793 804. 23.Graser,R.T.,T.P.DiLorenzo,F.Wang,G.J.Christianson, 7. Idoyaga, J., C. Fiorese, L. Zbytnuik, A. Lubkin, J. Miller, B. Malissen, H. D. Chapman, D. C. Roopenian, S. G. Nathenson, and D. V. Serreze. 2000. D. Mucida, M. Merad, and R. M. Steinman. 2013. Specialized role of Identification of a CD8 T cell that can independently mediate auto- migratory dendritic cells in peripheral tolerance induction. J. Clin. immune diabetes development in the complete absence of CD4 T cell – Invest. 123: 844 854. helper functions. J. Immunol. 164: 3913–3918. 8. Mukherjee, G., A. Geliebter, J. Babad, P. Santamaria, D. V. Serreze, 24. Verdaguer, J., D. Schmidt, A. Amrani, B. Anderson, N. Averill, and G. J. Freeman, K. V. Tarbell, A. Sharpe, and T. P. DiLorenzo. 2013. P. Santamaria. 1997. Spontaneous autoimmune diabetes in monoclonal by guest on September 27, 2021 DEC-205-mediated antigen targeting to steady-state dendritic cells T cell nonobese diabetic mice. J. Exp. Med. 186: 1663–1676. induces deletion of diabetogenic CD8+ T cells independently of PD-1 25.Jarchum,I.,T.Takaki,andT.P.DiLorenzo.2008.Efficient cul- and PD-L1. Int. Immunol. 25: 651–660. ture of CD8(+) T cells from the islets of NOD mice and their use for 9. Mukhopadhaya, A., T. Hanafusa, I. Jarchum, Y. G. Chen, Y. Iwai, the study of autoreactive specificities. J. Immunol. Methods 339: D. V. Serreze, R. M. Steinman, K. V. Tarbell, and T. P. DiLorenzo. 66–73. 2008. Selective delivery of b cell antigen to dendritic cells in vivo leads to ¨ deletion and tolerance of autoreactive CD8+ T cells in NOD mice. 26.Yoshida,K.,T.Martin,K.Yamamoto,C.Dobbs,C.Munz, Proc. Natl. Acad. Sci. USA 105: 6374–6379. N. Kamikawaji, N. Nakano, H. G. Rammensee, T. Sasazuki, K. Haskins, and H. Kikutani. 2002. Evidence for shared recognition of a 10. Petzold, C., J. Riewaldt, T. Koenig, S. Schallenberg, and peptide ligand by a diverse panel of non-obese diabetic mice- K. Kretschmer. 2010. Dendritic cell-targeted pancreatic b-cell antigen + derived, islet-specific, diabetogenic T cell clones. Int. Immunol. 14: leads to conversion of self-reactive CD4( ) T cells into regulatory 1439–1447. T cells and promotes immunotolerance in NOD mice. Rev. Diabet. Stud. 7: 47–61. 27. Crawford, F., B. Stadinski, N. Jin, A. Michels, M. Nakayama, P. Pratt, fi 11. Price, J. D., C. Hotta-Iwamura, Y. Zhao, N. M. Beauchamp, and P. Marrack, G. Eisenbarth, and J. W. Kappler. 2011. Speci city and + K. V. Tarbell. 2015. DCIR2+ cDC2 DCs and Zbtb32 restore CD4+ detection of insulin-reactive CD4 T cells in type 1 diabetes in the T-cell tolerance and inhibit diabetes. Diabetes 64: 3521–3531. nonobese diabetic (NOD) mouse. Proc. Natl. Acad. Sci. USA 108: – 12. Ring, S., M. Maas, D. M. Nettelbeck, A. H. Enk, and K. Mahnke. 2013. 16729 16734. Targeting of autoantigens to DEC205+ dendritic cells in vivo sup- 28. Lieberman, S. M., A. M. Evans, B. Han, T. Takaki, Y. Vinnitskaya, presses experimental allergic encephalomyelitis in mice. J. Immunol. J.A.Caldwell,D.V.Serreze,J.Shabanowitz,D.F.Hunt,S.G.Nathenson, 191: 2938–2947. et al. 2003. Identification of the b cell antigen targeted by a prevalent + 13. Price, J. D., and K. V. Tarbell. 2015. The role of dendritic cell subsets population of pathogenic CD8 T cells in autoimmune diabetes. Proc. and innate immunity in the pathogenesis of type 1 diabetes and other Natl. Acad. Sci. USA 100: 8384–8388. autoimmune diseases. Front. Immunol. 6: 288. 29. Lieberman, S. M., T. Takaki, B. Han, P. Santamaria, D. V. Serreze, and 14.Dong,M.B.,M.J.Rahman,andK.V.Tarbell.2016.Flowcytometric T. P. DiLorenzo. 2004. Individual nonobese diabetic mice exhibit gating for spleen monocyte and DC subsets: differences in autoimmune unique patterns of CD8+ T cell reactivity to three islet antigens, in- NOD mice and with acute inflammation. J. Immunol. Methods 432: 4–12. cluding the newly identified widely expressed dystrophia myotonica 15. Dudziak, D., A. O. Kamphorst, G. F. Heidkamp, V. R. Buchholz, kinase. J. Immunol. 173: 6727–6734. C. Trumpfheller, S. Yamazaki, C. Cheong, K. Liu, H. W. Lee, C. G. Park, 30. Creusot, R. J., J. Postigo-Fernandez, and N. Teteloshvili. 2018. Altered et al. 2007. Differential antigen processing by dendritic cell subsets in function of antigen-presenting cells in type 1 diabetes: A challenge for vivo. Science 315: 107–111. antigen-specific immunotherapy? Diabetes 67: 1481–1494.

https://doi.org/10.4049/immunohorizons.1900014 252 PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES ImmunoHorizons

31. Mbongue, J. C., H. A. Nieves, T. W. Torrez, and W. H. Langridge. 48. Nakayama, M., K. McDaniel, L. Fitzgerald-Miller, C. Kiekhaefer, 2017. The role of dendritic cell maturation in the induction of insulin- J. K. Snell-Bergeon, H. W. Davidson, M. Rewers, L. Yu, P. Gottlieb, dependent diabetes mellitus. Front. Immunol. 8: 327. J. W. Kappler, and A. Michels. 2015. Regulatory vs. inflammatory cytokine 32. Sozzani, S., A. Del Prete, and D. Bosisio. 2017. Dendritic cell re- T-cell responses to mutated insulin peptides in healthy and type 1 cruitment and activation in autoimmunity. J. Autoimmun. 85: diabetic subjects. Proc. Natl. Acad. Sci. USA 112: 4429–4434. 126–140. 49. Yang, J., I. T. Chow, T. Sosinowski, N. Torres-Chinn, 33. Yadav, D., N. Hill, H. Yagita, M. Azuma, and N. Sarvetnick. 2009. C. J. Greenbaum, E. A. James, J. W. Kappler, H. W. Davidson, and Altered availability of PD-1/PD ligands is associated with the failure to W. W. Kwok. 2014. Autoreactive T cells specific for insulin B:11-23 control autoimmunity in NOD mice. Cell. Immunol. 258: 161–171. recognize a low-affinity peptide register in human subjects with auto- 34. Lee, C. N., A. M. Lew, K. Shortman, and L. Wu. 2015. NOD mice are immune diabetes. Proc. Natl. Acad. Sci. USA 111: 14840–14845. functionally deficient in the capacity of cross-presentation. Immunol. 50. Christianson, S. W., L. D. Shultz, and E. H. Leiter. 1993. Adoptive Cell Biol. 93: 548–557. transfer of diabetes into immunodeficient NOD-scid/scid mice. Rela- + + 35. Rahman, M. J., G. Rahir, M. B. Dong, Y. Zhao, K. B. Rodrigues, tive contributions of CD4 and CD8 T-cells from diabetic versus a C. Hotta-Iwamura, Y. Chen, A. Guerrero, and K. V. Tarbell. 2016. prediabetic NOD.NON-Thy-1 donors. Diabetes 42: 44–55. Despite increased type 1 IFN, autoimmune nonobese diabetic mice 51.Price,J.D.,N.M.Beauchamp,G.Rahir,Y.Zhao,C.C.Rieger, display impaired dendritic cell response to CpG and decreased A. W. Lau-Kilby, and K. V. Tarbell. 2014. CD8+ dendritic cell-mediated + nuclear localization of IFN-activated STAT1. J. Immunol. 196: tolerance of autoreactive CD4 T cells is deficient in NOD mice and can Downloaded from 2031–2040. be corrected by blocking CD40L. J. Leukoc. Biol. 95: 325–336. 36. Chow, K. V., R. M. Sutherland, Y. Zhan, and A. M. Lew. 2017. Het- 52.Cohn,L.,B.Chatterjee,F.Esselborn,A.Smed-Sorensen,¨ erogeneity, functional specialization and differentiation of monocyte- N. Nakamura, C. Chalouni, B. C. Lee, R. Vandlen, T. Keler, P. Lauer, et al. derived dendritic cells. Immunol. Cell Biol. 95: 244–251. 2013. Antigen delivery to early endosomes eliminates the superiority of + 37. Jakubzick, C. V., G. J. Randolph, and P. M. Henson. 2017. Monocyte human blood BDCA3 dendritic cells at cross presentation. J. Exp. differentiation and antigen-presenting functions. Nat. Rev. Immunol. Med. 210: 1049–1063. 17: 349–362. 53. Chatterjee, B., A. Smed-Sorensen,¨ L. Cohn, C. Chalouni, R. Vandlen, http://www.immunohorizons.org/ 38. Bennett, C. L., and B. E. Clausen. 2007. DC ablation in mice: promises, B. C. Lee, J. Widger, T. Keler, L. Delamarre, and I. Mellman. 2012. pitfalls, and challenges. Trends Immunol. 28: 525–531. Internalization and endosomal degradation of receptor-bound anti- ffi 39. Hume, D. A. 2011. Applications of myeloid-specific promoters in gens regulate the e ciency of cross presentation by human dendritic – transgenic mice support in vivo imaging and functional genomics but cells. Blood 120: 2011 2020. do not support the concept of distinct macrophage and dendritic cell 54. Zerbino, D. R., P. Achuthan, W. Akanni, M. R. Amode, D. Barrell, lineages or roles in immunity. J. Leukoc. Biol. 89: 525–538. J. Bhai, K. Billis, C. Cummins, A. Gall, C. G. Giron,´ et al. 2018. Ensembl – 40. Qualai, J., L. X. Li, J. Cantero, A. Tarrats, M. A. Fernandez,´ L. Sumoy, 2018. Nucleic Acids Res. 46(D1): D754 D761. A. Rodolosse, S. J. McSorley, and M. Genesca.` 2016. Expression of 55.Fife,B.T.,I.Guleria,M.GubbelsBupp,T.N.Eagar,Q.Tang, CD11c is associated with unconventional activated T cell subsets with H.Bour-Jordan,H.Yagita,M.Azuma,M.H.Sayegh,andJ.A.Bluestone. high migratory potential. PLoS One 11: e0154253. 2006. Insulin-induced remission in new-onset NOD mice is main- tained by the PD-1-PD-L1 pathway. J. Exp. Med. 203: 2737–2747.

41. Zhou, V., K. Agle, X. Chen, A. Beres, R. Komorowski, L. Belle, by guest on September 27, 2021 C. Taylor, F. Zhu, D. Haribhai, C. B. Williams, et al. 2016. A colitogenic 56. Todd, J. A. 2010. Etiology of type 1 diabetes. Immunity 32: 457–467. memory CD4+ T cell population mediates gastrointestinal graft- 57. Vafiadis, P., S. T. Bennett, J. A. Todd, J. Nadeau, R. Grabs, versus-host disease. J. Clin. Invest. 126: 3541–3555. C. G. Goodyer, S. Wickramasinghe, E. Colle, and C. Polychronakos. 1997. 42. Jaeckel, E., M. A. Lipes, and H. von Boehmer. 2004. Recessive tol- Insulin expression in human thymus is modulated by INS VNTR al- erance to preproinsulin 2 reduces but does not abolish type 1 diabetes. leles at the IDDM2 locus. Nat. Genet. 15: 289–292. [Published errata appear in 2004 Nat. Immunol. 5: 1190; 2005 Nat. 58. Pugliese, A., M. Zeller, A. Fernandez Jr., L. J. Zalcberg, R. J. Bartlett, Immunol. 6: 219.] Nat. Immunol. 5: 1028–1035. C. Ricordi, M. Pietropaolo, G. S. Eisenbarth, S. T. Bennett, and 43. Krishnamurthy, B., N. L. Dudek, M. D. McKenzie, A. W. Purcell, D. D. Patel. 1997. The insulin gene is transcribed in the human thymus A. G. Brooks, S. Gellert, P. G. Colman, L. C. Harrison, A. M. Lew, and transcription levels correlated with allelic variation at the INS H. E. Thomas, and T. W. Kay. 2006. Responses against islet antigens VNTR-IDDM2 susceptibility locus for type 1 diabetes. Nat. Genet. 15: in NOD mice are prevented by tolerance to proinsulin but not IGRP. 293–297. J. Clin. Invest. 116: 3258–3265. 59. Arif, S., T. I. Tree, T. P. Astill, J. M. Tremble, A. J. Bishop, 44. Moriyama, H., N. Abiru, J. Paronen, K. Sikora, E. Liu, D. Miao, C. M. Dayan, B. O. Roep, and M. Peakman. 2004. Autoreactive T cell re- D. Devendra, J. Beilke, R. Gianani, R. G. Gill, and G. S. Eisenbarth. 2003. sponses show proinflammatory polarization in diabetes but a regu- Evidence for a primary islet autoantigen (preproinsulin 1) for insulitis latory phenotype in health. J. Clin. Invest. 113: 451–463. and diabetes in the nonobese diabetic mouse. Proc. Natl. Acad. Sci. USA 60. Kent, S. C., Y. Chen, L. Bregoli, S. M. Clemmings, N. S. Kenyon, 100: 10376–10381. C. Ricordi, B. J. Hering, and D. A. Hafler. 2005. Expanded T cells from 45. Nakayama, M., N. Abiru, H. Moriyama, N. Babaya, E. Liu, D. Miao, pancreatic lymph nodes of type 1 diabetic subjects recognize an in- L. Yu, D. R. Wegmann, J. C. Hutton, J. F. Elliott, and G. S. Eisenbarth. sulin epitope. Nature 435: 224–228. 2005. Prime role for an insulin epitope in the development of type 1 61. Mannering, S. I., L. C. Harrison, N. A. Williamson, J. S. Morris, diabetes in NOD mice. Nature 435: 220–223. D.J.Thearle,K.P.Jensen,T.W.Kay,J.Rossjohn,B.A.Falk,G.T.Nepom, 46. Daniel, D., R. G. Gill, N. Schloot, and D. Wegmann. 1995. Epitope and A. W. Purcell. 2005. The insulin A-chain epitope recognized by specificity, cytokine production profile and diabetogenic activity of human T cells is posttranslationally modified. J. Exp. Med. 202: insulin-specific T cell clones isolated from NOD mice. Eur. 1191–1197. J. Immunol. 25: 1056–1062. 62. Toma, A., S. Haddouk, J. P. Briand, L. Camoin, H. Gahery, F. Connan, 47. Alleva, D. G., P. D. Crowe, L. Jin, W. W. Kwok, N. Ling, M. Gottschalk, D. Dubois-Laforgue, S. Caillat-Zucman, J. G. Guillet, J. C. Carel, et al. P. J. Conlon, P. A. Gottlieb, A. L. Putnam, and A. Gaur. 2001. A 2005. Recognition of a subregion of human proinsulin by class I- disease-associated cellular immune response in type 1 diabetics to an restricted T cells in type 1 diabetic patients. Proc. Natl. Acad. Sci. USA immunodominant epitope of insulin. J. Clin. Invest. 107: 173–180. 102: 10581–10586.

https://doi.org/10.4049/immunohorizons.1900014 ImmunoHorizons PRECLINICAL MODEL FOR DENDRITIC CELL–TARGETED THERAPIES 253

63. Durinovic-Bello,´ I., R. P. Wu, V. H. Gersuk, S. Sanda, H. G. Shilling, 65. Selden, R. F., M. J. Skoskiewicz,´ K. B. Howie, P. S. Russell, and and G. T. Nepom. 2010. Insulin gene VNTR genotype associates with H. M. Goodman. 1986. Regulation of human insulin gene expression frequency and phenotype of the autoimmune response to proinsulin. in transgenic mice. Nature 321: 525–528. Genes Immun. 11: 188–193. 66. Dhodapkar, M. V., M. Sznol, B. Zhao, D. Wang, R. D. Carvajal, 64. Wong, F. S., J. Karttunen, C. Dumont, L. Wen, I. Visintin, I. M. Pilip, M.L.Keohan,E.Chuang,R.E.Sanborn,J.Lutzky,J.Powderly,etal.2014. N. Shastri, E. G. Pamer, and C. A. Janeway Jr. 1999. Identification of Induction of antigen-specific immunity with a vaccine targeting an MHC class I-restricted autoantigen in type 1 diabetes by screening NY-ESO-1 to the dendritic cell receptor DEC-205. Sci. Transl. Med. 6: an organ-specific cDNA library. Nat. Med. 5: 1026–1031. 232ra51. Downloaded from http://www.immunohorizons.org/ by guest on September 27, 2021

https://doi.org/10.4049/immunohorizons.1900014