It has become increasingly appreciated that autoimmune responses against neuronal components play an important role in type 1 diabetes (T1D) pathogenesis. In fact, a large proportion of islet-infiltrating B lymphocytes in the NOD mouse model of T1D produce Abs directed against the neuronal type III intermediate filament protein peripherin. NOD-PerIg mice are a previously developed BCR-transgenic model in which virtually all B lymphocytes express the H and L chain Ig molecules from the intra-islet–derived anti-peripherin–reactive hybridoma H280. NOD-PerIg mice have accelerated T1D development, and PerIg B lymphocytes actively proliferate within islets and expand cognitively interactive pathogenic T cells from a pool of naive precursors. We now report adoptively transferred T cells or whole splenocytes from NOD-PerIg mice expectedly induce T1D in NOD.scid recipients but, depending on the kinetics of disease development, can also elicit a peripheral neuritis (with secondary myositis). This neuritis was predominantly composed of CD4+ and CD8+ T cells. Ab depletion studies showed neuritis still developed in the absence of NOD-PerIg CD8+ T cells but required CD4+ T cells. Surprisingly, sciatic nerve–infiltrating CD4+ cells had an expansion of IFN-γ and TNF-α double-negative cells compared with those within both islets and spleen. Nerve and islet-infiltrating CD4+ T cells also differed by expression patterns of CD95, PD-1, and Tim-3. Further studies found transitory early B lymphocyte depletion delayed T1D onset in a portion of NOD-PerIg mice, allowing them to survive long enough to develop neuritis outside of the transfer setting. Together, this study presents a new model of peripherin-reactive B lymphocyte–dependent autoimmune neuritis.

The NOD mouse has greatly expanded our knowledge of the genetics and pathological underpinnings of autoimmune-mediated type 1 diabetes (T1D). However, NOD mice are also prone to the development of other autoimmune disorders, such as spontaneous salivary and lacrimal gland lesions reminiscent of Sjögren syndrome, spontaneous thyroiditis and parathyroiditis, and aging-associated neuritis/meningitis (1, 2). NOD mice are also susceptible to inducible diseases with clinical similarities to Hashimoto thyroiditis, systemic lupus erythematosus, myositis, colitis, and allergic encephalomyelitis (1, 2). Several genetic or pharmacological manipulations of NOD mice have also triggered the development of autoimmune neuritis. B7-2–deficient NOD mice spontaneously developed a peripheral neuritis, resulting in peripheral nerve demyelination and hind limb paralysis (3, 4). In this model, CD4+ T cells and IFN-γ were indispensable for disease development (3, 4), with the majority of pathogenic effectors specifically recognizing myelin protein 0 (5). ICAM-1–deficient NOD mice also develop a demyelinating form of neuritis mediated by CD4+ T cells, and although IFN-γ was increased in infiltrating T cells, IL-17 production determined disease severity (6). NOD-H2b/b Pdcd1−/− mice and, to a lesser extent, NOD-H2b/g7 Pdcd1−/− mice, which are both PD-1 deficient, also develop spontaneous CD4+ T cell–mediated neuritis (7). Despite the development of anti-myelin autoantibodies, this model does not require B lymphocyte help because genetic introduction of an Igμ−/− mutation did not abrogate neuritis development. Finally, IL-2 blockade in NOD mice, in addition to exacerbating T1D development, triggers the formation of multisystem autoimmunity, including peripheral neuropathy mediated both by CD4+ and, to a lesser extent, CD4 T cells (8).

Autoreactive T and B lymphocytes that recognize nervous system components are contributors to the natural course of T1D development in NOD mice. The well-known BDC2.5 CD4+ T cell clone originally isolated from the spleen of a diabetic NOD mouse (9) was eventually determined to respond to the neuronal as well as β cell–expressed protein chromogranin A (10) and thereafter to modified or hybrid versions thereof (1113). Abs against glutamic acid decarboxylase 65 (14) expressed in both neuronal and β cells have long been known as a marker for T1D risk in humans. Autoimmune responses mediating peri-islet neuronal Schwann cell death has been reported to precede β cell destruction in NOD mice (15, 16). Interestingly, a large number of hybridomas developed from islet-infiltrating B lymphocytes in NOD mice produce Abs against neuronal elements (17). The target Ag was later identified as peripherin (18). Abs directed against phosphorylated peripherin have also been detected in sera from a majority of tested T1D patients (19).

Peripherin is expressed during early development of β cells, although it is undetectable in adult pancreata (20). Accordingly, in adult NOD mice, anti-peripherin Abs stained neuronal components and not β cells (17). However, it has been hypothesized that early pancreatic immune infiltration during the timeframe in which islet expression of neuronal components occurs (21, 22) may cause cytokine-mediated increases in peripherin expression (23, 24). This could trigger an autoimmune response against peripherin during the early stages of T1D development (18). As a resource to test these possibilities, we created a NOD mouse stock (25) transgenically expressing the H and/or L chain Ig molecules from the peripherin-autoreactive hybridoma clone H280 (18, 26). Carriers of the H280 H (NOD-PerH) or L chain (NOD-PerL) alone both had accelerated T1D onset compared with standard NOD controls, and disease development was even more rapid in mice expressing both transgenes (NOD-PerIg) (25). NOD-PerIg B lymphocytes actively proliferate within the pancreatic islets (25). T cells from NOD.IgHEL.Igμnull mice (in which all B lymphocytes recognize the diabetes-irrelevant Ag hen egg lysozyme and thus cannot participate in the prior activation of diabetogenic T cells) transferred T1D more rapidly to NOD.scid-PerIg than standard NOD.scid recipients (25). This further indicated a role of peripherin-autoreactive B lymphocytes in expanding diabetogenic T cell responses. We initially aimed to learn more about the peripherin-reactive T cells that are expanded in NOD-PerIg mice. Surprisingly, we discovered that in addition to displaying diabetogenic activity T cells from young NOD-PerIg but not standard NOD mice can transfer an autoimmune neuritis (with secondary myositis) to NOD.scid recipients. This study assessed the pathogenic basis of the development of such neuritis.

NOD/ShiLtDvs (27) (hereafter NOD) and all other described mouse strains are maintained at The Jackson Laboratory under specific pathogen–free conditions. The NOD.Cg-Emv30b Prkdcscid/Dvs (hereafter NOD.scid) strain has been previously described (28). NOD/ShiLtDvs-Tg(IghH280)48Dvs Tg(IgkH280)934Dvs/Dvs (hereafter NOD-PerIg) and NOD.Cg-Emv30b Prkdcscid Tg(IghH280)48Dvs Tg(IgkH280)934Dvs/Dvs (hereafter NOD.scid-PerIg) mice transgenically expressing the H and L chains from peripherin-reactive hybridoma clone H280 (18, 26) have been previously described (25). Female mice were used for all experiments (donors and recipients). All mouse work has been approved by The Jackson Laboratory’s Animal Care and Use Committee.

Total T cell enrichment was accomplished via negative selection over LD columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Biotinylated B220-, CD11b-, and CD11c-specific Abs (Tonbo Biosciences, San Diego, CA) were used in conjunction with streptavidin MACS beads (Miltenyi Biotec) to negatively select away unwanted splenocyte populations. For T cell comixtures, additional biotinylated CD4- or CD8-specific Abs (BD Biosciences, San Jose, CA) were used to negatively select away the T cell subpopulation of choice.

CD8+ T cell depletion was accomplished by injecting 250 μg of anti-CD8 (53-6.72, originally from American Type Culture Collection, expanded and purified in house) i.p. every 3 wk. CD4+ T cell depletion was accomplished by injecting 250 μg of anti-CD4 (GK1.5, BE0003-1; Bio X Cell, West Lebanon, NH) i.p. weekly for the first four injections, followed by injections every 2 wk for the remainder of the study. Anti-CD8 and anti-CD4 injections were initiated in NOD.scid recipients on the same day they were engrafted with splenocytes. B lymphocyte depletion was accomplished by injecting i.p. 250 μg of anti-CD20 (MB20-11, IgG2C; MedImmune, Gaithersburg, MD) every 2 wk.

Mice were checked weekly for presence of glucosuria using Ames Diastix (Bayer, Leverkusen, Germany). Two readings of ≥0.25% (corresponding to ≥300 mg/dl in blood) on separate days was defined as T1D.

For all flow cytometry experiments, single-cell suspensions were prepared, and data were collected on either a FACSymphony A5, a BD FACSCalibur, or an LSR II SORP (all instruments from BD Biosciences). Data were analyzed using FlowJo Version 9/10 (BD Biosciences). Gey buffer was used to lyse RBCs for all spleen samples (29). For sciatic nerve preparations, nerves were removed from both hind limbs and placed in Dulbecco PBS with calcium and magnesium (Thermo Fisher Scientific, Waltham, MA). Nerves were transferred into 1 ml of digestion mixture containing HBSS (MilliporeSigma, St. Louis, MO), 5 U dispase (Collaborative Research, Waltham, MA) and 400 U collagenase D (MilliporeSigma). Nerves were mechanically disrupted, incubated for 30 min at 37°C, then triturated up and down. Digestion was inhibited with HBSS plus 2% FBS. The cell mixture was passed through 70-μm nytex to remove any remaining clumps. Hand picking of dissociated islets via a collagenase/DNase I digestion procedure has been previously described (30). The following modifications were performed: after the second pick into fresh media, islets were mechanically disrupted by vigorous pipetting.

For splenic engraftment experiments, samples were run on a BD FACSCalibur. No singlet discrimination was performed. Therefore, initial forward scatter (FSC)–height versus propidium iodide (PI) gating was used followed by gating on either CD4 or CD8 versus TCRβ among live cells, or CD19 among live cells followed by IgMa or IgMb gating. For sciatic nerve cellular subsets analysis assessed on a FACSymphony A5 or LSR II SORP, initial “rough leukocyte” FSC-area versus side scatter (SSC)–area gating was performed to exclude debris and small events with large SSC. This was followed by singlet discrimination performed via both FSC-area versus FSC-height and SSC-area versus SSC-height gating on the diagonal singlet cell populations. PI gating was performed to identify live cells, followed by gating on CD45.1 (see Supplemental Fig. 1). For cytokine and cell surface marker experiments assessed on a FACSymphony A5, initial gating started with the two singlet gates as described in Supplemental Fig. 1, followed by gating tightly on either Ghost Dye UV 450 (for cytokines; Tonbo Biosciences) or PI (for surface markers) versus CD45.1+ cells. T cell subset gating followed as shown in Fig. 3.

For intracellular cytokine staining, single-cell suspensions were plated in RPMI 1640 (Thermo Fisher Scientific) supplemented with 1× GlutaMAX (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), 1× MEM Non-Essential Amino Acids (Thermo Fisher Scientific), 100 μM 2-ME (Thermo Fisher Scientific), 100 U/ml penicillin (MilliporeSigma), 100 μg/ml streptomycin (MilliporeSigma), and 10% (v/v) heat-inactivated HyClone FBS (GE Healthcare Life Sciences, Marlborough, MA). Cells were initially stimulated for 5 h in the presence of 25 ng/ml PMA (MilliporeSigma), 1 μg/ml ionomycin (Cayman Chemical, Ann Arbor, MI), and GolgiPlug/brefeldin A (BD Biosciences) at the manufacturer’s recommended 1:1000 dilution. Extracellular markers were stained as normal. Cells were washed with Dulbecco PBS, then incubated with Ghost Dye UV 450 for live–dead discrimination. Cells were then fixed with Cytofix/Cytoperm (BD Biosciences) per the manufacturer’s recommended protocol. Intracellular staining was done in the presence of BD Perm/Wash Buffer (BD Biosciences).

Fluorochrome-conjugated mAbs were purchased from the following vendors: from BD Biosciences, Ly-6g (RB6-8C5; BV421), IgMb (AF6-78; PE), IgMa (DS-1; FITC), TCRβ (H57-597; BV711 or FITC), CD8α (53-6.7; BV480 or allophycocyanin), CD11b (M1/70; BV650), B220 (RA3-6B2; BUV496), CD45.1 (A20; FITC), Ly-6c (HK1.4; BV570), IL-4 (11B11; PE), IL-5 (TRFK5; PE), IL-17A (TC11-18H10, BV786), CD154/CD40L (MR1; PE), CD95/Fas (Jo2; FITC), and CD69 (H1.2F3; BUV737); from BioLegend (San Diego, CA), IgMa (MA-69; PE), CD90.2 (53.21; PE-Cy7), CD90.2 (30-H12; allophycocyanin-Cy7), CD4 (GK1.5; BV785), CD4 (RM4-5; BV570), IFN-γ (XMG1.2; allophycocyanin), TNF-α (MP6-XT22; PE-Cy7), TCRβ (H57-597; PE), CD223/LAG-3 (C9B7W; BV421), CD134/OX-40 (OX-86; PerCP-Cy5.5), CD366/TIM-3 (RMT3-23; PE-Cy7), CD357/GITR (DTA-1; PerCP-Cy5.5), CD278/ICOS (C398.4A; BV605), CD127/IL-7Rα (A7R34; BV650), and CD279/PD-1 (29F.1A12; BV711, CD25 (PC61; BV785); from Tonbo Biosciences, CD11c (N418; allophycocyanin), CD19 (ID3; RF710, allophycocyanin), and CD45.1 (A20; allophycocyanin); and from Thermo Fisher Scientific, CD4 (GK1.5, PE), IL-15 (eBio13A; PE), and CD178/FasL (MFL3; Super Bright 600).

Pancreata were fixed in Bouin solution (Rowley Biochemical, Danvers, MA) overnight and embedded in paraffin blocks. Three levels were cut and stained with H&E and aldehyde fuchsin. For neuritis studies, cohort 1 (Fig. 1A) mice were provided to and processed by The Jackson Laboratory Necropsy Core to determine the cause of the visible hind leg weakness. Subsequently, we focused on analysis of hind limbs for neuritis development. Briefly, hind limbs were fixed in 10% neutral buffered formalin (StatLab Medical Products, McKinney, TX), then transferred into ImmunoCal (StatLab Medical Products) for 48 h. Hind limbs provided the following sections: one hind limb provided two femur and tibia cross sections; the other hind limb provided two longitudinal (sagittal) sections. All sections were stained with H&E. Descriptive pathology details were provided by a blinded pathologist (R. Doty). In some figures, this descriptive pathology was converted to a number scale for purposes of plotting quantitative differences between groups. Specifics are provided in the figure legends. Limb, cranial, and spinal H&E histology images were obtained with an Olympus DP72 microscope digital camera using cellSens Standard 1.5 imaging software (Olympus, Tokyo, Japan). All nonsupplemental H&E histology images were processed in the following manner using GIMP 2.8.16 (GIMP Development Team; https://www.gimp.org): The images were scaled down to 2.1 inches wide, set at 300 dots per inch (to decrease figure file size), and, finally, vertically cropped to a final dimension of 2.1 × 1.5 inches in height. Cropping was performed to remove the embedded magnification/scale information, which was too small and pixelated to read at final publication size/resolution. Higher resolution scale information was superimposed on top of the cropped images in Inkscape 0.91 (Inkscape Project; https://inkscape.org/) during figure preparation.

Nerve histology has been described previously (31). Briefly, sciatic nerves were dissected free and fixed by immersion in 2% paraformaldehyde/2% glutaraldehyde in 0.1 M/l cacodylate buffer. Nerves were then plastic embedded, sectioned at 0.5 μm thickness, and stained with toluidine blue. Images were collected at 40× or 100× magnification on a Nikon Eclipse 600 microscope with DIC-Nomarski optics. Three blinded observers (T.J. Hines, A.L.D. Tadenev, and R.W. Burgess) examined the histology slides to assign the mice to control, clinical symptoms, and no-clinical-symptom groups based solely on histology.

Mean insulitis scores (MIS) were determined by a blinded observer (D.V. Serreze) using a previously described method (32). Briefly, for nondiabetic mice, aldehyde fuchsin–stained islets were scored at end of incidence as follows: no lesions, 0; peri-insular aggregates, 1; <25% islet destruction, 2; >25% islet destruction, 3; >75% islet destruction, 4. The final score was determined by dividing the total score for each pancreas by total islets examined.

Briefly, mice were anesthetized and placed on a heating pad. Legs were straightened and fixed in place with tape, then the ground recording electrode was placed s.c. in the left hind paw. The negative recording electrode was placed between the last two toes of the right hind paw, and the positive electrode was placed s.c. in the right hind paw. Stimulating electrodes were positioned at the ankle so that the two electrodes flanked the sciatic nerve. Beginning with 1 mA, the stimulus was increased until either a maximum compound muscle action potential (CMAP) amplitude, or a stimulus of 3 mA was first reached. The location of the stimulating electrodes was marked, and the electrodes were moved to the hip to stimulate at the sciatic notch using the same stimulus intensity. The distance between the two stimulation points, along with the latency for each stimulus to elicit an electromyographic response recorded in the foot, was used to determine the conduction velocity (m/s) (33).

All statistics (except for wheel running in Supplemental Fig. 3, which used the IBM SPSS Statistics 26 package; IBM, Armonk, NY) were calculated using Prism 7/8 (GraphPad, San Diego, CA). Scatter dot plots display bars indicating mean ± SEM where applicable. The p values for neuritis and flow cytometry scatter dot plots comparing two groups are two-tailed Mann–Whitney analyses. For graphs with two groups and two comparisons (Figs. 2B, 3H), a two-way ANOVA with Sidak multiple comparisons was performed. For graphs with three groups and one comparison, a one-way ANOVA with Tukey multiple comparisons was performed. For graphs with three groups and more than two comparisons (Fig. 5C, 5E), a two-way ANOVA with Tukey multiple comparison was performed. The p values for diabetes incidence curves are calculated by Mantel–Cox analysis.

We initially tested whether a dose of 1 × 106 purified T cells from five-week-old NOD-PerIg mice transferred T1D to NOD.scid recipients at a faster rate than T cells from NOD controls. Likely because of the young age of the donors, a single recipient of NOD T cells developed diabetes by 20 wk posttransfer. There was no T1D in recipients of NOD-PerIg T cells (Fig. 1A). However, during weekly glucosuria testing, it was observed that recipients of NOD-PerIg T cells began to display a sudden visible hind leg impairment after ∼15 wk posttransfer. Mice were sent to The Jackson Laboratory Necropsy Core to determine the cause of this visible impairment. Prosector observation prior to necropsy assessed the phenotype as uncoordinated hind leg movement while walking and an inability to discern presented edges. As detailed in Table I, blinded pathology results indicated control NOD.scid and NOD-PerIg mice had no visible lesions. Recipients of NOD T cells also had no visible lesions. However, recipients of NOD-PerIg T cells had severe lymphocytic neuritis in the hind limbs with some evidence of cranial and spinal nerve infiltration (Fig. 1B–D, Supplemental Fig. 2, Table I). Both mice examined for front limb involvement also had severe lymphocytic neuritis (Table I).

FIGURE 1.

NOD-PerIg T cells transfer diabetes as well as neuritis into NOD.scid recipients. (A) Diabetes incidence from cohort 1 NOD.scid recipients receiving 1 × 106 enriched T cells from five-week-old NOD or NOD-PerIg donors. Selected mice from cohort 1 were sent to necropsy for analysis of hind limbs, brain, and spine after hind limb weakness was observed. (B) Representative histology displaying signs of neuritis in a peripheral nerve of the hind limb (scale bar, 200 μm). (C) Representative image showing one cranial nerve (scale bar, 500 μm) that showed signs of infiltration. (D) Representative spinal nerve with signs of infiltration (scale bar, 500 μm). In (B)–(D), black arrows designate representative area of infiltration in affected tissue. (E) Diabetes incidence in cohort 2 NOD.scid recipients of 1 × 106 enriched T cells from seven-week-old NOD or NOD-PerIg donors. (F) Representative image of hind limb peripheral nerve neuritis in a cohort 2 recipient of NOD-PerIg T cells (scale bar, 200 μm). (G) Quantification of MIS for nondiabetic mice at 20 wk posttransfer comparing recipients of NOD or NOD-PerIg T cells.

FIGURE 1.

NOD-PerIg T cells transfer diabetes as well as neuritis into NOD.scid recipients. (A) Diabetes incidence from cohort 1 NOD.scid recipients receiving 1 × 106 enriched T cells from five-week-old NOD or NOD-PerIg donors. Selected mice from cohort 1 were sent to necropsy for analysis of hind limbs, brain, and spine after hind limb weakness was observed. (B) Representative histology displaying signs of neuritis in a peripheral nerve of the hind limb (scale bar, 200 μm). (C) Representative image showing one cranial nerve (scale bar, 500 μm) that showed signs of infiltration. (D) Representative spinal nerve with signs of infiltration (scale bar, 500 μm). In (B)–(D), black arrows designate representative area of infiltration in affected tissue. (E) Diabetes incidence in cohort 2 NOD.scid recipients of 1 × 106 enriched T cells from seven-week-old NOD or NOD-PerIg donors. (F) Representative image of hind limb peripheral nerve neuritis in a cohort 2 recipient of NOD-PerIg T cells (scale bar, 200 μm). (G) Quantification of MIS for nondiabetic mice at 20 wk posttransfer comparing recipients of NOD or NOD-PerIg T cells.

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Table I.
Histological findings in two cohorts of NOD-PerIg versus NOD T cell transfers
GroupTreatmentBrainSpineHind LimbFront Limb
Control NOD.scid (n = 3) None NVL NVL NVL  
Control NOD-PerIg (n = 3) None NVL NVL NVL  
NOD.scid (n = 4aNOD T Cells NVL NVL NVL  
NOD.scid (n = 7a, bPerIg T cells 3 of 5 mice examined: multifocal (few nerves affected). Primarily gliosis with few lymphocytes. 4 of 5 mice examined: multifocal (a few nerves affected), lymphocytic neuritis. 7 of 7 mice examined: severe lymphocytic neuritis. 2 of 2 mice examined: severe lymphocytic neuritis. 
NOD.scid (n = 10)c NOD T Cells   NVL  
NOD.scid (n = 13)c PerIg T cells   2 of 13 mice examined: lymphocytic neuritis.  
GroupTreatmentBrainSpineHind LimbFront Limb
Control NOD.scid (n = 3) None NVL NVL NVL  
Control NOD-PerIg (n = 3) None NVL NVL NVL  
NOD.scid (n = 4aNOD T Cells NVL NVL NVL  
NOD.scid (n = 7a, bPerIg T cells 3 of 5 mice examined: multifocal (few nerves affected). Primarily gliosis with few lymphocytes. 4 of 5 mice examined: multifocal (a few nerves affected), lymphocytic neuritis. 7 of 7 mice examined: severe lymphocytic neuritis. 2 of 2 mice examined: severe lymphocytic neuritis. 
NOD.scid (n = 10)c NOD T Cells   NVL  
NOD.scid (n = 13)c PerIg T cells   2 of 13 mice examined: lymphocytic neuritis.  
a

Cohort 1.

b

Six mice from cohort 1 (Fig. 1A) plus one mouse that had a thymoma at 15 wk and was removed from incidence study.

c

Cohort 2 (Fig. 1E, 1F).

A second cohort of NOD.scid mice were injected with 1 × 106 T cells enriched from seven-week-old NOD or NOD-PerIg donors to determine whether a similar phenotype was observed. This time, diabetes was efficiently transferred by both NOD and NOD-PerIg T cells, likely because of the slightly older age of the donors (Fig. 1E). In cohort 1, hind limb inflammation was more consistent than cranial and spinal nerve involvement (Supplemental Fig. 2, Table I). Thus, in cohort 2, hind limbs were analyzed for neuritis at diabetes onset or at the end of incidence. None of the recipients of NOD T cells had hind limb neuritis, whereas this phenotype was present in 2 of 13 of the recipients of NOD-PerIg T cells (Table I). Only three recipients of NOD-PerIg T cells in cohort 2 remained T1D free up to 15 wk posttransfer, when neuritis was present in similar cohort 1 recipients. One of these three mice subsequently developed T1D and had neuritis. Another such recipient also subsequently developed T1D without neuritis. The third recipient remained T1D free to 20 wk posttransfer and did develop neuritis (Fig. 1F). This mouse with neuritis but not T1D at 20 wk posttransfer had insulitis levels on par with the three recipients of NOD T cells that remained normoglycemic (Fig. 1G). These collective results indicate that neuritis is only mediated by T cells that previously had opportunities for interacting with peripherin-autoreactive B lymphocytes.

Next, we asked whether transfer of whole splenocytes from NOD-PerIg donors could also induce neuritis in NOD.scid or NOD.scid-PerIg recipients or whether neuritis development was a consequence of transferring purified T cell populations. Splenocytes from four- to six-week-old NOD-PerIg mice were transferred into NOD.scid or NOD.scid-PerIg recipients. T1D developed at a faster rate in NOD.scid-PerIg than NOD.scid recipients (Fig. 2A). Upon T1D development or at the end of the study, recipient spleens were harvested for evaluation of engraftment, pancreases were harvested for insulitis analysis, and hind limbs were examined for neuritis. As expected, NOD.scid-PerIg recipients had a large number of splenic PerIg B lymphocytes (mean 2.82 × 107) (Fig. 2B). These cells primarily represented the peripherin-autoreactive B lymphocytes endogenous to NOD.scid-PerIg recipients, with a minor number derived from the donor splenocyte inoculum. Much lower numbers (mean 9.65 × 105) of splenic B lymphocytes were found in NOD.scid recipients (Fig. 2B), all of which were derived from the donor mice. There was no difference in CD8+ T cell engraftment in the spleens of either recipient type (Fig. 2C). Conversely, there was a greater expansion of CD4+ T cells in the spleens of NOD.scid-PerIg recipients (Fig. 2D). Although none of the NOD.scid-PerIg recipients had any hind leg neuritis, four NOD.scid recipients had neuritis in both hind limbs, one had a single affected limb, and two were free of visible lesions (Fig. 2E). Thus, NOD-PerIg immunological effectors can only induce neuritis when transferred into completely lymphocyte-bereft NOD.scid recipients but not when transferred into NOD.scid-PerIg hosts in which peripherin-autoreactive B lymphocytes are present in large numbers. The ability of such peripherin-autoreactive B lymphocytes to quickly activate adoptively transferred diabetogenic T cells may not allow NOD.scid-PerIg hosts to survive long enough postengraftment to develop neuritis.

FIGURE 2.

NOD-PerIg whole splenocytes transfer neuritis into NOD.scid but not NOD.scid-PerIg recipients. Whole splenocytes (normalized to contain 1 × 106 T cells) from four- to six-week-old NOD-PerIg mice were transferred into NOD.scid or NOD.scid-PerIg recipients. (A) T1D incidence in NOD.scid and NOD.scid-PerIg recipients. (B) Quantification of yield of transgenic IgMa+ or nontransgenic IgMb+ CD19+ B cells in the spleens of NOD.scid and NOD.scid-PerIg recipients (NOD.scid, n = 7; NOD.scid-PerIg, n = 4). (C) Yield of CD8+ TCRβ+ cells in the spleens of NOD.scid and NOD.scid-PerIg recipients (NOD.scid, n = 7; NOD.scid-PerIg, n = 4). (D) Yield of CD4+ TCRβ+ cells in the spleens of NOD.scid and NOD.scid-PerIg recipients (NOD.scid, n = 7; NOD.scid-PerIg, n = 4). (E) Number of hind limbs displaying signs of neuritis (NOD.scid, n = 7; NOD.scid-PerIg, n = 10).

FIGURE 2.

NOD-PerIg whole splenocytes transfer neuritis into NOD.scid but not NOD.scid-PerIg recipients. Whole splenocytes (normalized to contain 1 × 106 T cells) from four- to six-week-old NOD-PerIg mice were transferred into NOD.scid or NOD.scid-PerIg recipients. (A) T1D incidence in NOD.scid and NOD.scid-PerIg recipients. (B) Quantification of yield of transgenic IgMa+ or nontransgenic IgMb+ CD19+ B cells in the spleens of NOD.scid and NOD.scid-PerIg recipients (NOD.scid, n = 7; NOD.scid-PerIg, n = 4). (C) Yield of CD8+ TCRβ+ cells in the spleens of NOD.scid and NOD.scid-PerIg recipients (NOD.scid, n = 7; NOD.scid-PerIg, n = 4). (D) Yield of CD4+ TCRβ+ cells in the spleens of NOD.scid and NOD.scid-PerIg recipients (NOD.scid, n = 7; NOD.scid-PerIg, n = 4). (E) Number of hind limbs displaying signs of neuritis (NOD.scid, n = 7; NOD.scid-PerIg, n = 10).

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We next assessed whether we could identify a means of predicting imminent neuritis development using measurable criteria. Toward this end, we generated another cohort of NOD.scid recipients engrafted with NOD-PerIg splenocytes and provided the mice to Jackson Laboratory’s Center for Biometric Analysis for blinded behavioral testing. Controls were NOD.scid mice injected with PBS. At week 15 posttransfer, NOD-PerIg recipients started to show symptoms of neuritis. Wheel running, rotarod, Von Frey, grip strength, and hotplate analyses were carried out up to 16 wk posttransfer. NOD-PerIg recipients displayed decreased balance and coordination in the rotarod assay and a modestly higher Von Frey response only at the 15–16 wk posttransfer time point, when neuritis was becoming overt (Supplemental Fig. 3). Only at this late postengraftment time point were NOD-PerIg recipients significantly less active during the night, as measured by voluntary running wheel activity (Supplemental Fig. 3E). Based on these data, it appears that prior to clinical symptoms, none of the tested mouse behaviors could be used to predict imminent neuritis onset. This is likely because of the clinical disease state developing suddenly, precluding an ability to observe any prior behavioral deficit.

Because of the overall negative results of the above behavioral analyses, we used a different approach to analyze the kinetics of nerve damage in this model. We set up a new cohort of NOD.scid mice engrafted with splenocytes from four- to six-week-old NOD-PerIg donors. Untreated NOD.scid mice provided controls. Mice were monitored weekly for T1D or neuritis development (Fig. 3A). Prior to 15 wk posttransfer, mice with T1D but no clinical signs of neuritis were removed from incidence, and sciatic nerves were analyzed for cellular infiltration (Fig. 3D–K). After 15 wk posttransfer, upon development of T1D or neuritis or at the end of incidence, mice were analyzed for nerve conduction velocity, sciatic nerve histology, and sciatic nerve cellular infiltration via flow cytometry (Fig. 3B–K). We should note, three mice with neuritis, one without clinical symptoms, and all surviving controls from the behavioral analysis cohort are also included in the below analyses (Fig. 3B–K).

FIGURE 3.

Nerve damage and immune cell infiltration. Whole splenocytes (normalized to contain 1 × 106 T cells) from four- to six-week-old NOD-PerIg mice were injected i.v. into NOD.scid recipients. (A) Mice were monitored weekly for T1D and/or neuritis development out to 21 wk posttransfer. At study initiation, 23 recipients of NOD-PerIg splenocytes were subsequently monitored for T1D/neuritis development. Any recipients developing T1D were removed from the study. Controls consisted of eight NOD.scid mice. Mice from this cohort, in addition to surviving mice from the behavioral studies, were analyzed for nerve damage (B and C) and immune cell infiltration (DJ). Mice from this cohort that developed T1D prior to 15 wk posttransfer were only subjected to immune cell infiltration analyses. (B) Quantification of hip CMAP comparing age-matched controls (unmanipulated or PBS-injected controls from behavioral cohort) to recipients of NOD-PerIg splenocytes. Mice with no detectable electromyography amplitudes have CMAP values of 0 mV. (C) Original magnification ×40 images of sciatic nerves comparing age-matched controls (left) to recipients of NOD-PerIg splenocytes with clinical symptoms of neuritis (center) or no disease symptoms (right). (D) Original magnification ×100 image of a sciatic nerve of a mouse with clinical symptoms of neuritis showing various indicators of pathology. Arrows: demyelinated axons; squares: myelin ovoids; circle: onion bulb. (E) Quantification of gated live cells showing percentage of leukocytes (CD45.1+) within sciatic nerves. (F and G) Representative flow cytometry plot (F) and quantification (G) showing the percentage of TCRβ+ CD90+ T cells among CD45.1+ cells within sciatic nerves. (H and I) Representative flow cytometry plots (H) and quantification (I) of gated T cells showing percentage of CD4+ versus CD8+ T cells within sciatic nerves. (J and K) Representative flow cytometry plots (J) and quantification (K) of gated leukocytes (CD45.1+) showing B220+ CD19+ B lymphocytes.

FIGURE 3.

Nerve damage and immune cell infiltration. Whole splenocytes (normalized to contain 1 × 106 T cells) from four- to six-week-old NOD-PerIg mice were injected i.v. into NOD.scid recipients. (A) Mice were monitored weekly for T1D and/or neuritis development out to 21 wk posttransfer. At study initiation, 23 recipients of NOD-PerIg splenocytes were subsequently monitored for T1D/neuritis development. Any recipients developing T1D were removed from the study. Controls consisted of eight NOD.scid mice. Mice from this cohort, in addition to surviving mice from the behavioral studies, were analyzed for nerve damage (B and C) and immune cell infiltration (DJ). Mice from this cohort that developed T1D prior to 15 wk posttransfer were only subjected to immune cell infiltration analyses. (B) Quantification of hip CMAP comparing age-matched controls (unmanipulated or PBS-injected controls from behavioral cohort) to recipients of NOD-PerIg splenocytes. Mice with no detectable electromyography amplitudes have CMAP values of 0 mV. (C) Original magnification ×40 images of sciatic nerves comparing age-matched controls (left) to recipients of NOD-PerIg splenocytes with clinical symptoms of neuritis (center) or no disease symptoms (right). (D) Original magnification ×100 image of a sciatic nerve of a mouse with clinical symptoms of neuritis showing various indicators of pathology. Arrows: demyelinated axons; squares: myelin ovoids; circle: onion bulb. (E) Quantification of gated live cells showing percentage of leukocytes (CD45.1+) within sciatic nerves. (F and G) Representative flow cytometry plot (F) and quantification (G) showing the percentage of TCRβ+ CD90+ T cells among CD45.1+ cells within sciatic nerves. (H and I) Representative flow cytometry plots (H) and quantification (I) of gated T cells showing percentage of CD4+ versus CD8+ T cells within sciatic nerves. (J and K) Representative flow cytometry plots (J) and quantification (K) of gated leukocytes (CD45.1+) showing B220+ CD19+ B lymphocytes.

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Although control NOD.scid mice had motor nerve conduction velocities of 26.72 ± 1.482 m/s, the recipients of NOD-PerIg splenocytes with visual signs of hind leg impairment had nearly undetectable responses, with only 4 of 11 producing readable CMAP amplitudes (Fig. 3B). Because of a lack of detectable CMAPs in 7 of 11 mice with clinical neuritis, nerve conduction velocities could only be calculated on four mice with clinical symptoms. Of the four clinical neuritis mice with detectable CMAP amplitudes, calculated nerve conduction velocities were similar to controls (22.86 ± 1.316 m/s). In contrast, all mice without visible signs of neuritis had CMAPs (Fig. 3B) and calculated motor nerve conduction velocities (22.36 ± 1.571 m/s) similar to controls. We should note, the large spread in CMAPs seen in control and no-clinical-neuritis mice is due to technical variation resulting from slight differences in electrode placement. However, it is important to reiterate that all control and no-clinical-neuritis mice had detectable CMAP amplitudes, whereas only 4 of 11 of those with clinical neuritis had such detectable responses. Sciatic nerve histology revealed that mice with clinical symptoms had hypercellularity and fewer axons. In the axons that did remain, there were signs of complete demyelination as well as the presence of myelin ovoids, indicative of ongoing demyelination (Fig. 3C, 3D). There were also occasional “onion bulb” structures, indicative of chronic demyelination and repair cycles (Fig. 3D). The combined loss of axons and demyelination of remaining axons likely explains the inability to generate detectable responses to nerve stimulation under the test conditions used (0.5 Hz, 1–3 mA) in a majority of recipients with overt neuritis (Fig. 3B). Recipients of NOD-PerIg splenocytes with no clinical indication of neuritis had nerve histology that was intermediate between controls and those with overt disease. Although some mice without clinical symptoms had histology that was indistinguishable from controls, others had signs of demyelination and increased interaxon-invasive cellularity compared with controls. For the most part, mice without clinical symptoms had nerves that were closer in appearance to controls than nerves from mice with overt neuritis (Fig. 3C). In fact, three blinded observers could not consistently assign a status of “control” or “no clinical symptoms” based solely on the appearance of nerve histology. Thus, these histological analyses also support an acute onset of disease pathology.

We prepared single-cell suspensions of sciatic nerves from NOD.scid controls or those engrafted with NOD-PerIg splenocytes to determine the makeup of infiltrating leukocyte populations. Mice with clinical symptoms of hind leg neuritis displayed a higher frequency of CD45.1+ leukocytes within sciatic nerves compared with NOD.scid controls (Fig. 3E, Supplemental Fig. 1E). Mice without clinical signs of neuritis had a much wider spread of CD45.1+ leukocytes within sciatic nerves. This is likely explained by the fact that some of these mice were likely near the cusp of developing clinical symptoms, whereas others were much earlier in the disease process. Among CD45.1+ cells, NOD.scid controls had little nonspecific background staining for CD90+ TCRβ+ cells, whereas ∼60% of leukocytes were T cells in recipients of NOD-PerIg splenocytes exhibiting overt neuritis (Fig. 3F, 3G). Mice without clinical signs of neuritis had decreased levels of T cells compared with those with overt disease; however, they had increased levels compared with NOD.scid controls (Fig. 3F, 3G). For all recipients of NOD.scid-PerIg splenocytes, infiltrating T cells were made up of both CD4+ and CD8+ T cells in a roughly 60:40 ratio (Fig. 3H, 3I). There was very little B lymphocyte infiltration (<4% of CD45.1+ cells) in recipients of NOD.scid-PerIg splenocytes (Fig. 3J, 3K). We also examined changes among CD45.1+ nonlymphocytes and found that, whereas there was an increase in CD11c+ cells and a decrease in Ly-6c+ cells among CD11b CD11c cells (Supplemental Fig. 1F), there were no other major differences in CD11b+ or CD11c+ subpopulations based off Ly-6c or Ly-6g gating (Supplemental Fig. 1G–I). Collectively, these data indicate the hind limb neuritis is, as expected, an overwhelmingly T cell–mediated disease, causing widespread damage to peripheral nerve architecture.

The CD4+ subset predominates T cells infiltrating sciatic nerves. However, we wondered whether neuritis transfer required both CD4+ and CD8+ T cells from NOD-PerIg mice or whether either population was sufficient to induce neuritis. To address these possibilities, we transferred NOD-PerIg splenocytes into NOD.scid recipients. Recipients were then treated with depleting CD8 or CD4 Abs. Controls were two separate cohorts of PBS vehicle-treated mice that were injected on the same schedule as those receiving anti-CD8 or anti-CD4 treatments. PBS mice are combined in the resulting analyses. It should be noted that the CD4 and CD8 Abs also served as reciprocal controls for each other. Although ∼60% of PBS-treated control recipients developed T1D by 25 wk posttransfer, none treated with either anti-CD8 or anti-CD4 did so (Fig. 4A). We analyzed sciatic nerves of mice upon either diabetes or clinical neuritis development or at the end of incidence for unafflicted mice. Among nondiabetic survivors, recipients treated with anti-CD8 or anti-CD4 had reduced insulitis scores compared with controls (Fig. 4B). Additionally, insulitis scores were lower in anti-CD4– than anti-CD8–treated mice (Fig. 4B). No differences were observed between PBS and anti-CD8–treated mice in terms of severity of neuritis (Fig. 4C, 4E, Table II). No anti-CD4–treated mice developed any clinical signs of neuritis, and only four mice had a few sporadic (i.e., nonclustered) lymphocytes in the sciatic nerves. This level of infiltration did not rise to the minimal neuritis designation observed in control or anti-CD8–treated mice (Fig. 4C). Interestingly, because clinical symptoms progressed slightly further than in previous experiments, some individuals among anti-CD8–injected mice and PBS-injected controls developed myositis (Fig. 4D, 4F, Table II). Anti-CD4–treated mice were completely free of myositis (Fig. 4D). Severity levels of neuritis and myositis in the NOD.scid recipients were not completely overlapping. However, no myositis was observed without at least a minimal level of co-occurring neuritis (Table II). Anti-CD4 or anti-CD8 treatment specifically removed their intended target populations (Fig. 4G, 4H). Anti-CD4–treated mice did have an expansion of splenic CD8+ T cells compared with control recipients (Fig. 4H). No differences were observed in splenic B lymphocyte engraftment (Fig. 4I).

FIGURE 4.

CD8+ and CD4+ T cell depletion prevents T1D, but only CD4+ depletion prevents neuritis development. Whole splenocytes from six-week-old NOD-PerIg donors (normalized to contain 1 × 106 T cells) were injected i.v. into NOD.scid recipients. One group of recipients were then injected i.p. every 3 wk with PBS or 250 μg of anti-CD8. Another group was injected i.p. once a week for 4 wk, followed by once every 2 wk with either PBS or 250 μg of anti-CD4. (A) T1D incidence in groups of NOD.scid recipients of NOD-PerIg splenocytes subsequently treated with PBS (both cohorts combined), anti-CD8, or anti-CD4. (B) MIS for nondiabetic survivors (n = 10 PBS, n = 13 anti-CD8, n = 13 anti-CD4). (C) Quantification of neuritis severity. Each dot represents a mouse with a pathology finding of severe, moderate, minimal, or no visible lesion (NVL) in hind leg pathology samples (n = 24 PBS, n = 13 anti-CD8, n = 13 anti-CD4). To display mean ± SEM, data were entered in Prism as severe = 3, moderate = 2, minimal = 1, NVL = 0, although the y-axis displays the pathologist’s description. For the anti-CD4 group, four mice had some sporadically spaced, nonclustered lymphocytes, technically indicating infiltration (because these are NOD.scid recipients). This infiltration, however, was closer in appearance to NVL than minimal but could not fully be classified as being free of lymphocyte infiltration. Therefore, for purposes of plotting, they were given a score of 0.25 to indicate that they were not free of infiltration but were closer to NVL than the representative minimal seen in (E). (D) Quantification of myositis severity. Each dot represents a mouse with a finding of myositis, minimal myositis, or no myositis (n = 24 PBS, n = 13 anti-CD8, n = 13 anti-CD4). To display mean ± SEM, data were entered in Prism as myositis = 2, minimal myositis = 1, no myositis = 0, although the y-axis displays the pathologist’s description. (E) Representative histology showing neuritis at NVL, minimal, and severe levels (scale bar, 200 μm). Black arrows designate representative area of infiltration in affected tissue. (F) Histology showing myositis at NVL, minimal, and severe levels (scale bar, 200 μm). For myositis, the most extreme case is shown. Black arrows designate a representative area of infiltration within affected tissue. (G) Quantification of CD4+ T cells in recipient spleens. (H) Quantification of CD8+ T cells in recipient spleens. (I) Quantification of CD19+ B lymphocytes in recipient spleens.

FIGURE 4.

CD8+ and CD4+ T cell depletion prevents T1D, but only CD4+ depletion prevents neuritis development. Whole splenocytes from six-week-old NOD-PerIg donors (normalized to contain 1 × 106 T cells) were injected i.v. into NOD.scid recipients. One group of recipients were then injected i.p. every 3 wk with PBS or 250 μg of anti-CD8. Another group was injected i.p. once a week for 4 wk, followed by once every 2 wk with either PBS or 250 μg of anti-CD4. (A) T1D incidence in groups of NOD.scid recipients of NOD-PerIg splenocytes subsequently treated with PBS (both cohorts combined), anti-CD8, or anti-CD4. (B) MIS for nondiabetic survivors (n = 10 PBS, n = 13 anti-CD8, n = 13 anti-CD4). (C) Quantification of neuritis severity. Each dot represents a mouse with a pathology finding of severe, moderate, minimal, or no visible lesion (NVL) in hind leg pathology samples (n = 24 PBS, n = 13 anti-CD8, n = 13 anti-CD4). To display mean ± SEM, data were entered in Prism as severe = 3, moderate = 2, minimal = 1, NVL = 0, although the y-axis displays the pathologist’s description. For the anti-CD4 group, four mice had some sporadically spaced, nonclustered lymphocytes, technically indicating infiltration (because these are NOD.scid recipients). This infiltration, however, was closer in appearance to NVL than minimal but could not fully be classified as being free of lymphocyte infiltration. Therefore, for purposes of plotting, they were given a score of 0.25 to indicate that they were not free of infiltration but were closer to NVL than the representative minimal seen in (E). (D) Quantification of myositis severity. Each dot represents a mouse with a finding of myositis, minimal myositis, or no myositis (n = 24 PBS, n = 13 anti-CD8, n = 13 anti-CD4). To display mean ± SEM, data were entered in Prism as myositis = 2, minimal myositis = 1, no myositis = 0, although the y-axis displays the pathologist’s description. (E) Representative histology showing neuritis at NVL, minimal, and severe levels (scale bar, 200 μm). Black arrows designate representative area of infiltration in affected tissue. (F) Histology showing myositis at NVL, minimal, and severe levels (scale bar, 200 μm). For myositis, the most extreme case is shown. Black arrows designate a representative area of infiltration within affected tissue. (G) Quantification of CD4+ T cells in recipient spleens. (H) Quantification of CD8+ T cells in recipient spleens. (I) Quantification of CD19+ B lymphocytes in recipient spleens.

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Table II.
Histological findings for mice injected every 3 wk with either PBS or anti-CD8
Mouse IdentificationTreatmentNeuritisMyositis
AF18748 PBS NVL No 
AF18749 PBS NVL No 
AF18750 PBS Minimal No 
AF18723 PBS Severe Yes 
AF18724 PBS Severe Yes 
AF18725 PBS Severe No 
AF18739 PBS NVL No 
AF18740 PBS Severe No 
AF18741 PBS Severe Yes 
AF18742 PBS Severe Yes 
AF18729 Anti-CD8 Severe Yes 
AF18732 Anti-CD8 Minimal Mild 
AF18606 Anti-CD8 Severe Yes 
AF18607 Anti-CD8 Severe Yes 
AF18608 Anti-CD8 Severe Yes 
AF18764 Anti-CD8 Minimal No 
AF18765 Anti-CD8 Minimal No 
AF18766 Anti-CD8 Minimal No 
AF18767 Anti-CD8 Moderate Mild 
AF18773 Anti-CD8 Mild No 
AF18774 Anti-CD8 NVL No 
AF18775 Anti-CD8 Severe No 
AF18776 Anti-CD8 Minimal No 
AF18777 Anti-CD8 Minimal No 
Mouse IdentificationTreatmentNeuritisMyositis
AF18748 PBS NVL No 
AF18749 PBS NVL No 
AF18750 PBS Minimal No 
AF18723 PBS Severe Yes 
AF18724 PBS Severe Yes 
AF18725 PBS Severe No 
AF18739 PBS NVL No 
AF18740 PBS Severe No 
AF18741 PBS Severe Yes 
AF18742 PBS Severe Yes 
AF18729 Anti-CD8 Severe Yes 
AF18732 Anti-CD8 Minimal Mild 
AF18606 Anti-CD8 Severe Yes 
AF18607 Anti-CD8 Severe Yes 
AF18608 Anti-CD8 Severe Yes 
AF18764 Anti-CD8 Minimal No 
AF18765 Anti-CD8 Minimal No 
AF18766 Anti-CD8 Minimal No 
AF18767 Anti-CD8 Moderate Mild 
AF18773 Anti-CD8 Mild No 
AF18774 Anti-CD8 NVL No 
AF18775 Anti-CD8 Severe No 
AF18776 Anti-CD8 Minimal No 
AF18777 Anti-CD8 Minimal No 

To begin addressing the potential mechanisms by which CD4+ T cells initiate nerve destruction, we set up three additional cohorts of NOD.scid recipients (Fig. 5A). In addition to analyzing sciatic nerve–infiltrating T cells, we also examined islet-infiltrating T cells in nondiabetic neuritic or unafflicted survivors. Both islet- and sciatic nerve–infiltrating CD4+ T cells had a small expansion of IFN-γ producing cells compared with those in spleens (Fig. 5B, 5C). In contrast, both sites of inflammation had reductions in TNF-α producing CD4+ T cells (Fig. 5B, 5C) compared with the spleen, with the greatest reduction being found in those within sciatic nerves. For all three tissues, the proportion of TNF-α–producing CD4+ T cells was greater than IFN-γ–producing cells, although the disparity was greater in the spleen and islets compared with the sciatic nerve (p < 0.0001 for spleen and islet, p = 0.0458 for sciatic nerve). Very little TH2 (IL-4, -5, and -13) or TH17 (IL-17) CD4+ T cells were observed (Fig. 5B, 5C). Taken together, these data indicate the potential contribution of IFN-γ and TNF-α in sciatic nerve damage.

FIGURE 5.

Islet- and sciatic nerve–infiltrating CD4+ T cells are highly activated. NOD.scid mice were injected with whole splenocytes (normalized to contain 1 × 106 T cells) from four to six-week-old NOD-PerIg mice. Mice were monitored weekly for T1D and neuritis development. Upon diabetes or neuritis development, mice were removed from incidence, and spleen- and sciatic nerve–infiltrating CD4+ T cells were examined for cytokine production and surface marker phenotype by flow cytometry. For nondiabetic mice, islet-infiltrating CD4+ T cells were also analyzed. (A) T1D or neuritis incidence in NOD.scid recipients of NOD-PerIg splenocytes (n = 38 from three separate cohorts). (B) Representative staining pattern of IFN-γ, IL-4, -5, -13, IL-17A, and TNF-α on live gated sciatic nerve CD45.1+ CD90+ TCRβ+ CD4+ cells. (C) Quantification showing mean ± SEM percentage indicated cytokine among CD4+ T cells in the indicated organ. Data are combined from five to six experiments (spleen, n = 12 for TNF-α and n = 21 for other cytokines; islet, n = 10 for TNF-α and n = 11 for other cytokines; sciatic nerve, n = 17 for TNF-α and n = 26 for other cytokines). (D) Representative staining pattern for IFN-γ versus TNF-α on live gated sciatic nerve CD45.1+ CD90+ TCRβ+ CD4+ cells. (E) Quantification of indicated cytokine combination among CD4+ T cells in the indicated organ. Data are combined from three to four experiments (spleen, n = 12; islet n = 10, sciatic nerve, n = 17). (F) Quantification of median fluorescence intensity (MFI) of the indicated cell surface marker on gated CD4+ T cells showing mean ± SEM. Data are combined from four to five experiments (spleen, n = 17; islet, n = 14; sciatic nerve, n = 21). (G) Based off CD4+CD25+ phenotype, proportion of regulatory T cells in spleens, islets, and sciatic nerves. Data are combined from four to five experiments (spleen, n = 17; islet, n = 14; sciatic nerve n = 21). (H) Quantification of MFI of the indicated cell surface marker on gated CD4+ T cells showing mean ± SEM. Data are combined from four to five experiments (spleen, n = 17; islet, n = 14; sciatic nerve, n = 21). (I) Representative staining pattern for CD4 versus TIM-3 on live gated sciatic nerve CD45.1+ CD90+ TCRβ+ CD4+ cells showing gated TIM-3HI cells. (J) Quantification of the proportion of TIM-3HI cells among CD4+ T cells. Data are combined from two to three experiments per organ (spleen, n = 10; islet, n = 11; sciatic nerve, n = 14).

FIGURE 5.

Islet- and sciatic nerve–infiltrating CD4+ T cells are highly activated. NOD.scid mice were injected with whole splenocytes (normalized to contain 1 × 106 T cells) from four to six-week-old NOD-PerIg mice. Mice were monitored weekly for T1D and neuritis development. Upon diabetes or neuritis development, mice were removed from incidence, and spleen- and sciatic nerve–infiltrating CD4+ T cells were examined for cytokine production and surface marker phenotype by flow cytometry. For nondiabetic mice, islet-infiltrating CD4+ T cells were also analyzed. (A) T1D or neuritis incidence in NOD.scid recipients of NOD-PerIg splenocytes (n = 38 from three separate cohorts). (B) Representative staining pattern of IFN-γ, IL-4, -5, -13, IL-17A, and TNF-α on live gated sciatic nerve CD45.1+ CD90+ TCRβ+ CD4+ cells. (C) Quantification showing mean ± SEM percentage indicated cytokine among CD4+ T cells in the indicated organ. Data are combined from five to six experiments (spleen, n = 12 for TNF-α and n = 21 for other cytokines; islet, n = 10 for TNF-α and n = 11 for other cytokines; sciatic nerve, n = 17 for TNF-α and n = 26 for other cytokines). (D) Representative staining pattern for IFN-γ versus TNF-α on live gated sciatic nerve CD45.1+ CD90+ TCRβ+ CD4+ cells. (E) Quantification of indicated cytokine combination among CD4+ T cells in the indicated organ. Data are combined from three to four experiments (spleen, n = 12; islet n = 10, sciatic nerve, n = 17). (F) Quantification of median fluorescence intensity (MFI) of the indicated cell surface marker on gated CD4+ T cells showing mean ± SEM. Data are combined from four to five experiments (spleen, n = 17; islet, n = 14; sciatic nerve, n = 21). (G) Based off CD4+CD25+ phenotype, proportion of regulatory T cells in spleens, islets, and sciatic nerves. Data are combined from four to five experiments (spleen, n = 17; islet, n = 14; sciatic nerve n = 21). (H) Quantification of MFI of the indicated cell surface marker on gated CD4+ T cells showing mean ± SEM. Data are combined from four to five experiments (spleen, n = 17; islet, n = 14; sciatic nerve, n = 21). (I) Representative staining pattern for CD4 versus TIM-3 on live gated sciatic nerve CD45.1+ CD90+ TCRβ+ CD4+ cells showing gated TIM-3HI cells. (J) Quantification of the proportion of TIM-3HI cells among CD4+ T cells. Data are combined from two to three experiments per organ (spleen, n = 10; islet, n = 11; sciatic nerve, n = 14).

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The above analyses did not account for coexpression of IFN-γ and TNF-α. Thus, we examined the extent these populations of IFN-γ– and TNF-α–expressing cells may overlap. No differences were observed in IFN-γ– and TNF-α–coexpressing cells, whereas there was a slight increase in IFN-γ+ TNF-α CD4+ cells in both islets and sciatic nerves compared with the spleen (Fig. 5D, 5E). There was a decrease in the proportion of IFN-γ TNF-α+ CD4+ T cells in islets, and an even greater reduction within sciatic nerves (Fig. 5D, 5E). Most significantly, both islets and sciatic nerve CD4+ T cells had an expansion of IFN-γ TNF-α cells (Fig. 5D, 5E). Taken together, the modest expansion of IFN-γ–producing cells and the drop in those producing TNF-α–producing cells may indicate a greater role for the former cytokine in sciatic nerve damage. However, the greatest change appears to be the expansion of IFN-γ TNF-α cells.

The expansion of IFN-γ TNF-α cells within sites of inflammation was surprising. To further dissect the nature of CD4+ T cells in the spleen and sites of inflammation, we next examined markers for cell surface activation (Fig. 5F) and tolerance (Fig. 5G–J). As expected, compared with those within spleens, CD4+ T cells infiltrating islets or sciatic nerves were highly activated, having increased expression of CD69, ICOS, and IL-7Rα (Fig. 5F). Furthermore, based on a CD4+CD25+ phenotype, proportions of regulatory T cells with a potential ability to modulate the activation state of effector populations did not differ in spleens, islets, and sciatic nerves of NOD.scid recipients engrafted with NOD-PerIg splenocytes (Fig. 5G). Taken together, these data indicate that despite the expansion of an IFN-γ TNF-α population in both sciatic nerves and islets, CD4+ T cells at these sites are more activated than their splenic counterparts.

Next, we examined markers of function and tolerance. We were unable to detect much expression of FasL, CD40L, OX40, or LAG-3 on CD4+ T cells (data not shown). However, islet-infiltrating CD4+ T cells had increased expression of CD95 compared with those within spleens and sciatic nerves (Fig. 5H). PD-1 expression was increased in sciatic nerve–infiltrating CD4+ T cells and to a greater extent by those within islets than that observed within spleens (Fig. 5H). Finally, we found that sciatic nerve–infiltrating CD4+ T cells had an expansion of TIM-3Hi cells compared with those in spleens and islets (Fig. 5I, 5J). Taken together, these data indicate that, whereas exhibiting some differences, islet- and sciatic nerve–infiltrating CD4+ cells do have high expression of negative costimulatory molecules, indicating they have progressed through the end stages of activation.

Finally, we asked whether the neuritis phenomenon was limited to transfer conditions or, if T1D could be attenuated, could this pathology eventually develop in NOD-PerIg mice. The accelerated T1D development in NOD-PerIg mice is due to peripherin-reactive B lymphocytes (25). Despite known issues of lessened efficacy of anti-CD20 in NOD mice (34, 35), we reasoned that even partial depletion of NOD-PerIg B lymphocytes from an early age might delay T1D development. Starting at four weeks of age, we treated NOD-PerIg mice with anti-CD20. Anti-CD20 treatment significantly delayed the time of disease onset but not the overall high penetrance of T1D in NOD-PerIg mice (Fig. 6A). One of two mice that developed T1D at 18 wk of age showed signs of neuritis (Fig. 6B). Two mice, taken down at 27 wk of age showed no signs of neuritis. A final mouse, taken down at 29 wk of age with no T1D, showed signs of both neuritis and myositis (Fig. 6C, 6D). Taken together, these data indicate NOD-PerIg mice are capable of developing neuritis, but their accelerated T1D status normally masks their susceptibility to this pathology. Additionally, these data indicate neuritis-inducing NOD T cells only require early transient interactions with peripherin-reactive B lymphocytes.

FIGURE 6.

B lymphocyte depletion reveals neuritis can develop in NOD-PerIg mice if T1D is delayed. NOD-PerIg mice were injected i.p with 250 μg of anti-CD20 every 2 wk starting at 4 wk of age. (A) T1D incidence comparing unmanipulated or anti-CD20–treated mice. (B) Representative histology of a diabetic mouse with neuritis. Black arrow designates representative area of infiltration in affected nerve tissue. (C and D) Mouse that survived to 29 wk of age without T1D, showing representative histology of (C) neuritis and (D) myositis. Black arrow designates representative area of infiltration in affected tissue.

FIGURE 6.

B lymphocyte depletion reveals neuritis can develop in NOD-PerIg mice if T1D is delayed. NOD-PerIg mice were injected i.p with 250 μg of anti-CD20 every 2 wk starting at 4 wk of age. (A) T1D incidence comparing unmanipulated or anti-CD20–treated mice. (B) Representative histology of a diabetic mouse with neuritis. Black arrow designates representative area of infiltration in affected nerve tissue. (C and D) Mouse that survived to 29 wk of age without T1D, showing representative histology of (C) neuritis and (D) myositis. Black arrow designates representative area of infiltration in affected tissue.

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To our knowledge, this is the first model of peripheral neuritis in NOD mice that is based off expansion of T cells mediated by T1D-contributory, peripherin-autoreactive B lymphocytes. Previous reports of peripheral neuritis in NOD mice have been associated with modulations to immune costimulation (4, 68) as opposed to our model, in which disease is initiated by a naturally occurring autoreactive B lymphocyte population. Although T1D can be transferred by T cells from NOD-PerIg mice, depending on the kinetics of that syndrome, antiperipheral nerve autoimmune responses can also occur. Our current findings also provide further evidence of the overlap in the autoimmune repertoire that targets islets versus nervous system components, as has been reported in T1D and multiple sclerosis patients (36).

We have several lines of evidence that peripherin-reactive B lymphocytes interact with cognate T cells in the NOD.PerIg mouse models. First, in NOD.PerIg mice, there is an increase in both germinal center B lymphocytes and T follicular helper cells compared with standard NOD controls (25). Second, naive T cells (NOD-IgHEL.Igμnull) transferred diabetes at a faster rate to NOD.scid-PerIg mice than to standard NOD.scid mice, and disease exacerbation was associated with an expansion of CD4+ T cells (25). Third, we know NOD-PerIg B lymphocytes actively participate within the islet lesion, as evidenced by their extensive proliferation at this site (25), which would require both cognate Ag and T cell help (25). And finally, preliminary studies have found that purified NOD-PerIg B lymphocytes engraft better in NOD.scid recipients when cotransferred with cognate NOD-PerIg but not NOD T cells (Supplemental Fig. 4).

In all previously reported cases of neuritis, nerves have undergone demyelination (4, 68), with myelin itself being identified as an antigenic target in several of these models (5, 6). Thus, these represent primary demyelinating models. In the peripheral nervous system, peripherin is a subunit of neurofilaments (37) within axons. The demyelination observed in this study is likely secondary to axon damage initiated by T cells that have been expanded by anti-peripherin but not polyclonal B lymphocytes. If peripherin remains the target Ag in the peripheral nerves, we propose two sites where peripherin-reactive T cells would likely initiate axonal destruction: unmyelinated neurons or nodes of Ranvier. However, the presence of unmyelinated axons might indicate primary reactivity against Schwann cells, and myelin and not peripherin itself. Because inflammatory cytokines can cause the formation of peripherin aggregates, which in turn initiates apoptosis of motor neurons (38), there also exists the possibility of early unmyelinated nerve damage caused by peripherin-reactive T cells triggering a cascade of surrounding neuronal death that could expand the immune response against other neuronal Ags. It is also possible that an immune response directed against myelinated axons at the exposed nodes of Ranvier leads to damage of the flanking Schwann cells triggering the demyelination observed in this study. An immune response targeting axonal Ags at the nodes of Ranvier would not be unprecedented, as recent evidence suggests that nodal regions are the target of B lymphocytes in Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy (39).

Few NOD-PerIg B lymphocytes are present within neuritis lesions despite the active role they play in insulitis. This, coupled with the fact that purified T cells from NOD-PerIg mice transfer neuritis without the need to cotransfer PerIg B cells, indicates these B cells play a minor role in the lesion after initiating the expansion of neuritis-causing T cells. The reason for the disparity between islet and nerve infiltration of NOD-PerIg B lymphocytes is currently unknown. Whether an initial anti-peripherin immune response in the islets (driven by the interaction between the peripherin-reactive PerIg B lymphocytes and cognate T cells) eventually expands anti-myelin (or other neuronal Ag) autoreactive cells is currently unknown. To address whether peripherin does remain the specific Ag being targeted in the peripheral nerves, work is currently ongoing to produce a direct-in-NOD CRISPR/Cas9 Prph1 knockout mouse. Because peripherin plays an important role in the development of unmyelinated neurons (40), this may provide its own set of complications.

As previously stated, our neuritis model relies on the expanded presence of a naturally occurring diabetogenic peripherin-autoreactive B lymphocyte population (25) and not changes to costimulatory signaling (4, 68). Although only two anti-CD20–treated NOD-PerIg mice developed neuritis (Fig. 6A), this is likely because, whereas exhibiting a somewhat slowed disease onset, they were still characterized by aggressive T1D development. We have not observed any clinical signs of neuritis development in standard NOD mice undergoing types of B lymphocyte deletion therapies (34, 35) out to 40 weeks of age. Furthermore, we are currently unaware of any reports of NOD mice given long term insulin injections to extend survival developing sudden-onset neuritis. However, there have been cases of age-related neuritis/meningitis development in unmanipulated NOD/ShiLtJ mice (1, 2) a close relation to our NOD substrain (NOD/ShiLtDvs) (27). We suggest that longer term preclinical T1D intervention studies in NOD mice may be required to determine whether other autoimmune diseases become more prominent when T1D is attenuated. T1D and multiple sclerosis patients reportedly have overlapping T cell repertoires with a potential to target both nervous systems components and islets (36). Thus, it is also possible that an intervention ultimately found to inhibit T1D development in humans at high risk for this disease might engender the appearance of other autoimmune pathologies in such individuals.

We have previously reported myositis development in NOD mice carrying a transgene encoding a CD2 promoter-driven IFN-γR β-chain (41). In that model, myositis appears to develop in the lumbar region of the spinal column prior to migrating to the limbs, without the appearance of neuritis. NOD-PerIg T cells appear to target peripheral nerves first, as we documented myositis development in both anti-CD8– and PBS-treated recipients when mice were not immediately removed from incidence studies upon initial visible neuritis onset. As they were not allowed to deteriorate further after the appearance of overt neuritis, it is unknown whether these mice would have subsequently developed a similar level of myositis characterizing the CD2 promoter-driven IFN-γR β-chain transgenic NOD stock (41). Furthermore, no myositis was observed without at least minimal neuritis (Table II). We did observe cases of severe neuritis without accompanying myositis (Table II). Whether the T cells that infiltrate the nerves are different from those causing myositis or whether the populations involved in the myositis in this study are similar to those observed in our previous study (41) is not currently known.

We were surprised by the expansion of IFN-γ TNF-α T cells in islets and sciatic nerves compared with the spleen (Fig. 5D, 5E). However, because of the highly activated nature of the cells in islets and sciatic nerves (Fig. 5F) and the expansion of negative-feedback costimulatory molecules (Fig. 5H–J), it is possible we are detecting the expansion of terminally differentiated cells that are no longer participating in disease pathogenesis. Interestingly, the pattern of these markers differs between islets and sciatic nerves (Fig. 5H–J), likely indicating these cells differ between the two sites of inflammation. The expansion of TIM-3HI cells in the sciatic nerves (Fig. 5I, 5J) may indicate a narrower range of Ag-reactive T cells than within islets, hence the progression to a more TIM-3+–exhausted phenotype.

Finally, the finding that CD8-depleted splenocytes from NOD-PerIg donors allowed development of neuritis but not T1D is interesting. The lack of T1D development resulting from CD8+ T cell depletion was expected, as such cells from standard NOD donors are required to transfer this disease to NOD.scid recipients, unless the donors are already hyperglycemic (42). However, the ability to transfer aggressive neuritis may indicate that sufficient antineuronal CD4+ T cell responses developed at an early age in NOD-PerIg donors, whereas young NOD-PerIg mice have not yet generated a repertoire of diabetogenic CD4+ T cells capable of independently inducing hyperglycemia in secondary recipients. We do not know how T cells infiltrating sciatic nerves may be related to those recognizing peripherin that attack pancreatic β cells. However, our cell surface marker analysis reveals a slightly different phenotype of CD4+ T cells in islets versus sciatic nerves, which may indicate a divergent pool of Ag-specific T cells. To fully dissect this issue, future studies identifying unique and overlapping TCR sequences between islet- and nerve-infiltrating T cells is warranted but is outside the scope of this initial report.

The authors are grateful to Rachael Pelletier for making the initial observations that lead to the discovery of neuritis in this model. We thank Drs. Jeffery Twiss (University of South Carolina) and Ahmet Hoke (Johns Hopkins School of Medicine) for comments on the description of peripheral nerve histopathology. We also thank The Jackson Laboratory Research Animal Facility, Flow Cytometry Core, Transgenic Genotyping Core, and Comparative Medicine and Quality Department, Center for Biometric Analysis, and the Histopathology Sciences group. Finally, the authors are grateful for the support of Carl Stiewe and his wife, Maike Rohde, whose generous donation toward T1D research at The Jackson Laboratory has contributed to our work.

J.J.R. is supported by Juvenile Diabetes Research Foundation (JDRF) Fellowship 3-PDF-2017-372-A-N. D.V.S. is supported by National Institutes of Health (NIH), National Institute of Diabetes and Digestive and Kidney Diseases Grants DK46266, DK95735, and NIH, Office of the Director Grant OD-020351, as well as by JDRF Grant 2018-568. R.W.B.’s work on this project was supported by NIH, National Institute of Neurological Disorders and Stroke Grants NS054154 and NIH, Office of the Director Grant OD-020351. This work was also partly supported by NIH, National Cancer Institute Grant CA34196.

The online version of this article contains supplemental material.

Abbreviations used in this article:

CMAP

compound muscle action potential

FSC

forward scatter

MIS

mean insulitis score

NVL

no visible lesion

PI

propidium iodide

SSC

side scatter

T1D

type 1 diabetes.

1
Leiter
,
E.
,
M.
Atkinson
.
1998
.
NOD Mice and Related Strains: Research Applications in Diabetes, AIDS, Cancer, and Other Diseases.
R.G. Landes
,
Austin, TX
.
2
Leiter
,
E. H.
1993
.
The NOD mouse: a model for analyzing the interplay between heredity and environment in development of autoimmune disease.
ILAR J.
35
:
4
14
.
3
Bour-Jordan
,
H.
,
H. L.
Thompson
,
J. A.
Bluestone
.
2005
.
Distinct effector mechanisms in the development of autoimmune neuropathy versus diabetes in nonobese diabetic mice.
J. Immunol.
175
:
5649
5655
.
4
Salomon
,
B.
,
L.
Rhee
,
H.
Bour-Jordan
,
H.
Hsin
,
A.
Montag
,
B.
Soliven
,
J.
Arcella
,
A. M.
Girvin
,
J.
Padilla
,
S. D.
Miller
,
J. A.
Bluestone
.
2001
.
Development of spontaneous autoimmune peripheral polyneuropathy in B7-2-deficient NOD mice. [Published erratum appears in 2001 J. Exp. Med. 194: 1393.]
J. Exp. Med.
194
:
677
684
.
5
Louvet
,
C.
,
B. G.
Kabre
,
D. W.
Davini
,
N.
Martinier
,
M. A.
Su
,
J. J.
DeVoss
,
W. L.
Rosenthal
,
M. S.
Anderson
,
H.
Bour-Jordan
,
J. A.
Bluestone
.
2009
.
A novel myelin P0-specific T cell receptor transgenic mouse develops a fulminant autoimmune peripheral neuropathy.
J. Exp. Med.
206
:
507
514
.
6
Meyer zu Horste
,
G.
,
A. K.
Mausberg
,
S.
Cordes
,
H.
El-Haddad
,
H. J.
Partke
,
V. I.
Leussink
,
M.
Roden
,
S.
Martin
,
L.
Steinman
,
H. P.
Hartung
,
B. C.
Kieseier
.
2014
.
Thymic epithelium determines a spontaneous chronic neuritis in Icam1(tm1Jcgr)NOD mice.
J. Immunol.
193
:
2678
2690
.
7
Yoshida
,
T.
,
F.
Jiang
,
T.
Honjo
,
T.
Okazaki
.
2008
.
PD-1 deficiency reveals various tissue-specific autoimmunity by H-2b and dose-dependent requirement of H-2g7 for diabetes in NOD mice.
Proc. Natl. Acad. Sci. USA
105
:
3533
3538
.
8
Setoguchi
,
R.
,
S.
Hori
,
T.
Takahashi
,
S.
Sakaguchi
.
2005
.
Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization.
J. Exp. Med.
201
:
723
735
.
9
Haskins
,
K.
,
M.
Portas
,
B.
Bradley
,
D.
Wegmann
,
K.
Lafferty
.
1988
.
T-lymphocyte clone specific for pancreatic islet antigen.
Diabetes
37
:
1444
1448
.
10
Stadinski
,
B. D.
,
T.
Delong
,
N.
Reisdorph
,
R.
Reisdorph
,
R. L.
Powell
,
M.
Armstrong
,
J. D.
Piganelli
,
G.
Barbour
,
B.
Bradley
,
F.
Crawford
, et al
.
2010
.
Chromogranin A is an autoantigen in type 1 diabetes.
Nat. Immunol.
11
:
225
231
.
11
Delong
,
T.
,
T. A.
Wiles
,
R. L.
Baker
,
B.
Bradley
,
G.
Barbour
,
R.
Reisdorph
,
M.
Armstrong
,
R. L.
Powell
,
N.
Reisdorph
,
N.
Kumar
, et al
.
2016
.
Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion.
Science
351
:
711
714
.
12
Delong
,
T.
,
R. L.
Baker
,
J.
He
,
K.
Haskins
.
2013
.
Novel autoantigens for diabetogenic CD4 T cells in autoimmune diabetes.
Immunol. Res.
55
:
167
172
.
13
Delong
,
T.
,
R. L.
Baker
,
J.
He
,
G.
Barbour
,
B.
Bradley
,
K.
Haskins
.
2012
.
Diabetogenic T-cell clones recognize an altered peptide of chromogranin A.
Diabetes
61
:
3239
3246
.
14
Baekkeskov
,
S.
,
H. J.
Aanstoot
,
S.
Christgau
,
A.
Reetz
,
M.
Solimena
,
M.
Cascalho
,
F.
Folli
,
H.
Richter-Olesen
,
P.
De Camilli
.
1990
.
Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. [Published erratum appears in 1990 Nature 347: 782.]
Nature
347
:
151
156
.
15
Tsui
,
H.
,
Y.
Chan
,
L.
Tang
,
S.
Winer
,
R. K.
Cheung
,
G.
Paltser
,
T.
Selvanantham
,
A. R.
Elford
,
J. R.
Ellis
,
D. J.
Becker
, et al
.
2008
.
Targeting of pancreatic glia in type 1 diabetes.
Diabetes
57
:
918
928
.
16
Winer
,
S.
,
H.
Tsui
,
A.
Lau
,
A.
Song
,
X.
Li
,
R. K.
Cheung
,
A.
Sampson
,
F.
Afifiyan
,
A.
Elford
,
G.
Jackowski
, et al
.
2003
.
Autoimmune islet destruction in spontaneous type 1 diabetes is not beta-cell exclusive.
Nat. Med.
9
:
198
205
.
17
Carrillo
,
J.
,
M. C.
Puertas
,
A.
Alba
,
R. M.
Ampudia
,
X.
Pastor
,
R.
Planas
,
N.
Riutort
,
N.
Alonso
,
R.
Pujol-Borrell
,
P.
Santamaria
, et al
.
2005
.
Islet-infiltrating B-cells in nonobese diabetic mice predominantly target nervous system elements.
Diabetes
54
:
69
77
.
18
Puertas
,
M. C.
,
J.
Carrillo
,
X.
Pastor
,
R. M.
Ampudia
,
R.
Planas
,
A.
Alba
,
R.
Bruno
,
R.
Pujol-Borrell
,
J. M.
Estanyol
,
M.
Vives-Pi
,
J.
Verdaguer
.
2007
.
Peripherin is a relevant neuroendocrine autoantigen recognized by islet-infiltrating B lymphocytes.
J. Immunol.
178
:
6533
6539
.
19
Doran
,
T. M.
,
J.
Morimoto
,
S.
Simanski
,
E. J.
Koesema
,
L. F.
Clark
,
K.
Pels
,
S. L.
Stoops
,
A.
Pugliese
,
J. S.
Skyler
,
T.
Kodadek
.
2016
.
Discovery of phosphorylated peripherin as a major humoral autoantigen in type 1 diabetes mellitus.
Cell Chem. Biol.
23
:
618
628
.
20
Escurat
,
M.
,
K.
Djabali
,
C.
Huc
,
F.
Landon
,
C.
Bécourt
,
C.
Boitard
,
F.
Gros
,
M.-M.
Portier
.
1991
.
Origin of the beta cells of the islets of Langerhans is further questioned by the expression of neuronal intermediate filament proteins, peripherin and NF-L, in the rat insulinoma RIN5F cell line.
Dev. Neurosci.
13
:
424
432
.
21
Hauben
,
E.
,
M. G.
Roncarolo
,
U.
Nevo
,
M.
Schwartz
.
2005
.
Beneficial autoimmunity in type 1 diabetes mellitus.
Trends Immunol.
26
:
248
253
.
22
Saravia
,
F.
,
F.
Homo-Delarche
.
2003
.
Is innervation an early target in autoimmune diabetes?
Trends Immunol.
24
:
574
579
.
23
Sterneck
,
E.
,
D. R.
Kaplan
,
P. F.
Johnson
.
1996
.
Interleukin-6 induces expression of peripherin and cooperates with Trk receptor signaling to promote neuronal differentiation in PC12 cells.
J. Neurochem.
67
:
1365
1374
.
24
Troy
,
C. M.
,
N. A.
Muma
,
L. A.
Greene
,
D. L.
Price
,
M. L.
Shelanski
.
1990
.
Regulation of peripherin and neurofilament expression in regenerating rat motor neurons.
Brain Res.
529
:
232
238
.
25
Leeth
,
C. M.
,
J.
Racine
,
H. D.
Chapman
,
B.
Arpa
,
J.
Carrillo
,
J.
Carrascal
,
Q.
Wang
,
J.
Ratiu
,
L.
Egia-Mendikute
,
E.
Rosell-Mases
, et al
.
2016
.
B-lymphocytes expressing an Ig specificity recognizing the pancreatic ß-cell autoantigen peripherin are potent contributors to type 1 diabetes development in NOD mice.
Diabetes
65
:
1977
1987
.
26
Carrillo
,
J.
,
M. C.
Puertas
,
R.
Planas
,
X.
Pastor
,
A.
Alba
,
T.
Stratmann
,
R.
Pujol-Borrell
,
R. M.
Ampudia
,
M.
Vives-Pi
,
J.
Verdaguer
.
2008
.
Anti-peripherin B lymphocytes are positively selected during diabetogenesis.
Mol. Immunol.
45
:
3152
3162
.
27
Simecek
,
P.
,
G. A.
Churchill
,
H.
Yang
,
L. B.
Rowe
,
L.
Herberg
,
D. V.
Serreze
,
E. H.
Leiter
.
2015
.
Genetic analysis of substrain divergence in non-obese diabetic (NOD) mice.
G3 (Bethesda)
5
:
771
775
.
28
Serreze
,
D. V.
,
E. H.
Leiter
,
M. S.
Hanson
,
S. W.
Christianson
,
L. D.
Shultz
,
R. M.
Hesselton
,
D. L.
Greiner
.
1995
.
Emv30null NOD-scid mice. An improved host for adoptive transfer of autoimmune diabetes and growth of human lymphohematopoietic cells.
Diabetes
44
:
1392
1398
.
29
Julius
,
M. H.
,
L. A.
Herzenberg
.
1974
.
Isolation of antigen-binding cells from unprimed mice: demonstration of antibody-forming cell precursor activity and correlation between precursor and secreted antibody avidities.
J. Exp. Med.
140
:
904
920
.
30
Serreze
,
D. V.
,
E. H.
Leiter
,
S. M.
Worthen
,
L. D.
Shultz
.
1988
.
NOD marrow stem cells adoptively transfer diabetes to resistant (NOD x NON)F1 mice.
Diabetes
37
:
252
255
.
31
Seburn
,
K. L.
,
K. H.
Morelli
,
A.
Jordanova
,
R. W.
Burgess
.
2014
.
Lack of neuropathy-related phenotypes in hint1 knockout mice.
J. Neuropathol. Exp. Neurol.
73
:
693
701
.
32
Johnson
,
E. A.
,
P.
Silveira
,
H. D.
Chapman
,
E. H.
Leiter
,
D. V.
Serreze
.
2001
.
Inhibition of autoimmune diabetes in nonobese diabetic mice by transgenic restoration of H2-E MHC class II expression: additive, but unequal, involvement of multiple APC subtypes.
J. Immunol.
167
:
2404
2410
.
33
Burgess
,
R. W.
,
G. A.
Cox
,
K. L.
Seburn
.
2010
.
Neuromuscular disease models and analysis.
Methods Mol. Biol.
602
:
347
393
.
34
Wang
,
Q.
,
J. J.
Racine
,
J. J.
Ratiu
,
S.
Wang
,
R.
Ettinger
,
C.
Wasserfall
,
M. A.
Atkinson
,
D. V.
Serreze
.
2017
.
Transient BAFF blockade inhibits type 1 diabetes development in nonobese diabetic mice by enriching immunoregulatory B lymphocytes sensitive to deletion by anti-CD20 cotherapy.
J. Immunol.
199
:
3757
3770
.
35
Serreze
,
D. V.
,
H. D.
Chapman
,
M.
Niens
,
R.
Dunn
,
M. R.
Kehry
,
J. P.
Driver
,
M.
Haller
,
C.
Wasserfall
,
M. A.
Atkinson
.
2011
.
Loss of intra-islet CD20 expression may complicate efficacy of B-cell-directed type 1 diabetes therapies.
Diabetes
60
:
2914
2921
.
36
Winer
,
S.
,
I.
Astsaturov
,
R.
Cheung
,
L.
Gunaratnam
,
V.
Kubiak
,
M. A.
Cortez
,
M.
Moscarello
,
P. W.
O’Connor
,
C.
McKerlie
,
D. J.
Becker
,
H. M.
Dosch
.
2001
.
Type I diabetes and multiple sclerosis patients target islet plus central nervous system autoantigens; nonimmunized nonobese diabetic mice can develop autoimmune encephalitis.
J. Immunol.
166
:
2831
2841
.
37
Yuan
,
A.
,
T.
Sasaki
,
A.
Kumar
,
C. M.
Peterhoff
,
M. V.
Rao
,
R. K.
Liem
,
J. P.
Julien
,
R. A.
Nixon
.
2012
.
Peripherin is a subunit of peripheral nerve neurofilaments: implications for differential vulnerability of CNS and peripheral nervous system axons.
J. Neurosci.
32
:
8501
8508
.
38
Robertson
,
J.
,
J. M.
Beaulieu
,
M. M.
Doroudchi
,
H. D.
Durham
,
J. P.
Julien
,
W. E.
Mushynski
.
2001
.
Apoptotic death of neurons exhibiting peripherin aggregates is mediated by the proinflammatory cytokine tumor necrosis factor-alpha.
J. Cell Biol.
155
:
217
226
.
39
Devaux
,
J. J.
,
M.
Odaka
,
N.
Yuki
.
2012
.
Nodal proteins are target antigens in Guillain-Barré syndrome.
J. Peripher. Nerv. Syst.
17
:
62
71
.
40
Larivière
,
R. C.
,
M. D.
Nguyen
,
A.
Ribeiro-da-Silva
,
J. P.
Julien
.
2002
.
Reduced number of unmyelinated sensory axons in peripherin null mice.
J. Neurochem.
81
:
525
532
.
41
Serreze
,
D. V.
,
M. A.
Pierce
,
C. M.
Post
,
H. D.
Chapman
,
H.
Savage
,
R. T.
Bronson
,
P. B.
Rothman
,
G. A.
Cox
.
2003
.
Paralytic autoimmune myositis develops in nonobese diabetic mice made Th1 cytokine-deficient by expression of an IFN-gamma receptor beta-chain transgene.
J. Immunol.
170
:
2742
2749
.
42
DiLorenzo
,
T. P.
,
R. T.
Graser
,
T.
Ono
,
G. J.
Christianson
,
H. D.
Chapman
,
D. C.
Roopenian
,
S. G.
Nathenson
,
D. V.
Serreze
.
1998
.
Major histocompatibility complex class I-restricted T cells are required for all but the end stages of diabetes development in nonobese diabetic mice and use a prevalent T cell receptor alpha chain gene rearrangement.
Proc. Natl. Acad. Sci. USA
95
:
12538
12543
.

R.E. was a former employee of MedImmune and holds stock in AstraZeneca. She is currently an employee and shareholder of Viela Bio. The other authors have no financial conflicts of interest.

Supplementary data