Subcutaneous insulin B:9-23/IFA immunisation induces Tregs that control late-stage prediabetes in NOD mice through IL-10 and IFNγ
- 1.2k Downloads
Subcutaneous immunisation with the 9–23 amino acid region of the insulin B chain (B:9-23) in incomplete Freund’s adjuvant (IFA) can protect the majority of 4- to 6-week-old prediabetic NOD mice and is currently in clinical trials. Here we analysed the effect of B:9-23/IFA immunisation at later stages of the disease and the underlying mechanisms.
NOD mice were immunised once s.c. with B:9-23/IFA at 5 or 9 weeks of age, or when blood glucose reached 10 mmol/l or higher. Diabetes incidence was followed in addition to variables such as regulatory T cell (Treg) induction, cytokine production (analysed by Elispot) and emergence of pathogenic CD8+/NRP-V7+ cells.
A single B:9-23/IFA immunisation protected the majority of NOD mice at advanced stages of insulitis, but not after blood glucose reached 13.9 mmol/l. It increased Treg numbers and lost its protective effect after IFNγ or IL-10 neutralisation, but not in the absence of IL-4. CD4+CD25+ and to a lesser extent IFNγ-producing cells from mice protected by B:9-23/IFA induced tolerance upon transfer into new NOD animals, indicating that a dominant Treg-mediated effect was operational. Reduced numbers of CD8+/NRP-V7+ memory T cells coincided with protection from the disease.
Protection from diabetes after B:9-23/IFA immunisation cannot be achieved once diabetes is fully established, but can be achieved at most prediabetic stages of the disease. Protection is mediated by Tregs that require IFNγ and IL-10. These findings should provide important guidance for ongoing human trials, especially for the development of suitable T cell biomarkers.
KeywordsB:9-23 IFNγ IL-4 IL-10 Incomplete Freund’s adjuvant Tregs Type 1 diabetes
9-23 Amino acid region of the insulin B chain
Cytotoxic T lymphocyte antigen 4
Forkhead box p3
Intracellular cytokine staining
Incomplete Freund’s adjuvant
Islet-specific glucose-6-phosphatase catalytic subunit-related protein
Pancreatic draining lymph nodes
Type 1 diabetes is one of the most common autoimmune diseases affecting the lives of millions of patients, among them children. As with most autoimmune diseases, many different autoantigens can be involved in the pathogenic response against the insulin-producing beta cells of the pancreas. In humans and mice, islet autoantibody specificities and epitopes for CD4+ and CD8+ T cells recognising insulin, GAD, islet tyrosine phosphatase IA-2 and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) have been identified [1, 2, 3, 4], but it is unclear which of these autoantigens are crucial for diabetes development. In NOD mice, the 9–23 amino acid region of the insulin B chain (B:9-23) is the dominant epitope involved in initiation of the disease [5, 6, 7]. In light of recent advances, various beta cell-specific therapeutic approaches have been developed and tested in animal models of diabetes, primarily in NOD mice . Due to their specific immune-modulating effects, antigen-specific approaches represent preferable avenues for immune intervention .
Antigen-specific therapies can be applied before or after diabetes onset, depending on their efficacy [8, 9, 10, 11]. The goal of beta cell antigen-specific approaches is to ‘re-educate’ the immune system to stop it attacking the beta cells of the pancreas [9, 12, 13]. A large panel of autoantigenic epitopes has been established for humans and mice. Insulin-based interventions have shown very promising results in mice [14, 15, 16, 17] and based on these findings, human clinical trials with insulin are currently on the way . Prevention of diabetes following immunisation with insulin or insulin-derived epitopes has succeeded in many experimental settings, e.g. after intramuscular DNA vaccination, after oral treatment, or after intranasal and s.c. peptide vaccination in the presence or absence of enhancers such as incomplete Freund’s adjuvant (IFA). For B:9-23 immunisation, most of these approaches have shown protection from diabetes, with the precise mechanism being unknown [14, 15, 16, 17, 19, 20, 21, 22]. The great importance of better defining the underlying mechanisms is that this could provide a basis for defining biomarkers that would help monitor the effect of B:9-23 immunisation on the progression of beta cell autoimmunity in clinical trials.
With respect to s.c. B:9-23/IFA immunisation, all previously published studies were conducted at a rather early stage of the disease in the NOD mouse (4–6 weeks old) [14, 15]. We reasoned that it would be important to carry out additional B:9-23/IFA immunisation studies at a later, more advanced stage of the disease (9 weeks old and recent-onset) in order to better reflect the human scenario, where prediabetic patients with different degrees of beta cell loss and targeted autoantigens are being recruited. Of great importance for current clinical trials currently implementing B:9-23/IFA immunisation after diabetes onset is our finding that this intervention may not be beneficial in patients with advanced stages of the disease, unless significant residual beta cell mass is present. Investigation of the underlying immunological mechanisms revealed enhancement of IFNγ, IL-10 and to lesser extent IL-4 production by Tregs in the spleen and the pancreatic draining lymph nodes (PDLN). Interestingly, IFNγ and IL-10, but not IL-4 were required for the protective effect. Both populations, Tregs (CD4+CD25+ cells) and IFNγ-producing cells, possessed long- or short-term regulatory properties respectively, demonstrating that a dominant regulatory effect was operational. In summary, we report here that B:9-23/IFA immunisation operates through Tregs that produce IFNγ and IL-10, and is an effective immune intervention strategy with the ability to halt diabetes progression at advanced stages of insulitis, but not long after disease is established.
NOD/LtJ and NOD/IL-4 −/− female mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). All mice were maintained at La Jolla Institute for Allergy and Immunology animal facility under pathogen-free conditions and handled in accordance with protocols approved by the organisation’s Animal Care and Use Committee.
Blood glucose monitoring
Blood glucose was monitored twice a week with a monitoring system (OneTouch Ultra; LifeScan, Milpitas, CA, USA). Diabetes was defined as two consecutive blood glucose values above 13.9 mmol/l.
Definition of treatment groups
Mice with blood glucose <10 mmol/l at 4 or 9 weeks of age were characterised as prediabetic stages I and II respectively. Mice older than 10 weeks of age with blood glucose 10 to 13.9 mmol/l at first reading were defined as prediabetic stage III and those with blood glucose 13.9 to 19.4 mmol/l as recent-onset diabetes. Mice with blood glucose 19.4 to 33.3 mmol/l were characterised as established diabetes.
B:9-23 peptide treatments
B:9-23 peptide (amino acid sequence: SHLVEALYLVCGERG) was purchased from Αbgent (San Diego, CA, USA) with more than 95% wt/wt purity. After dissolving in DMSO/DPBS, it was emulsified in IFA (1:1) at 0.5 mg/ml. Injections of 200 µl (100 µg) were performed s.c. at the neck area. This was done once in each mouse at either 5 or 9 weeks of age, or when blood glucose exceeded 10 mmol/l. In control groups, mice were treated with DMSO/PBS/IFA or left untreated.
After a 2.4 G2 blocking step, cells were stained for CD4-PacificBlue CD8α-APCCy7 (BD-Pharmingen, San Diego, CA, USA), and CD25-FITC and CD127-PeCy7 (eBioscience, San Diego, CA, USA). For intracellular forkhead box p3 (FOXP3) detection, cells were fixed with Fix/Perm buffer and stained with FOXP3-antigen-presenting cell (APC; eBioscience). For intracellular cytokine staining (ICCS), cells were initially surface-stained with CD4-PerCP5.5, CD25-APCCy7 and CD8a-PECy7, and then fixed and stained for IL-10-APC (BD-Pharmingen) and IFNγ-Pacific Blue (eBioscience) using a kit (Cytofix/Cytoperm; BD Biosciences) according to manufacturers’ instructions. For NRP-V7 staining, cells were stained with 1:100 NRP-V7-PE tetramer  at room temperature for 30 min. An antibody cocktail containing CD44-PacificBlue (Biolegend, San Diego, CA, USA), and CD4-PerCP5.5, CD19-FITC and CD8-APC (BD-Pharmingen) was added and another incubation step followed. All antibody incubations were performed at 4°C for 30 min (isotype controls were included). Cells were immediately acquired on a flow cytometer (LSRII; BD Biosciences) and analysed using a software package (FlowJo; Treestar, Ashland, OR, USA).
Assessment of cytokine production by single lymphoid cells
All antibodies against IL-10, IL-4 and IFNγ were from BD-Pharmingen except those for IL-17 (eBioscience). Briefly, 96-well millititer HA plates (Millipore, Bedford, MA, USA) were coated with capture antibodies at 5 µg/ml. After a 10% (vol./vol.) FCS/HL-1 blocking step, CD8-depleted (clone 53-6.7; BD-Pharmingen) splenocyte suspensions were added at various dilutions ranging from 1 × 106 to 0.125 × 106 cells/well and cultured in 2.5% (vol./vol.) FCS/HL-1 medium. Cells from blood or PDLN were plated at 0.25 × 106 cells/well. T cell-depleted splenocytes were added as APCs from age-matched non-treated NOD mice at a 1:1 ratio. B:9-23 peptide (10 µg/ml) was used in the presence of rhIL-2 (50 U/ml). After 2 days incubation at 37°C and extensive washes, detection antibodies were added at 4 µg/ml in PBS/Tween/1% (vol./vol.) FCS. Following an avidin-peroxidase step (1:1,000 dilution; Vector Laboratories, Burlingame, CA, USA,) colour was developed with 3-amino-9-ethylcarbazole (Sigma-Aldrich, St Louis, MO, USA) and H2O2. Finally, plates were rinsed in water, left to air-dry and spots representing cytokine-producing cells were counted using computer-assisted image analysis (Elispot reader; Carl Zeiss MicroImaging, Thornwood, NY, USA).
Pancreases were immersed in Tissue-Tek OCT (Bayer, Torrance, CA, USA), quick-frozen and cut into 6 µm tissue sections. After sections were fixed in 100% vol./vol. acetone and dried, they were re-hydrated in TBS and an avidin/biotin-blocking step was included (Vector Laboratories). To detect insulin and CD4 or CD8 in pancreatic sections, primary antibodies were applied as follows: guinea pig anti-swine insulin (1:300; Dako, Carpinteria, CA, USA), and anti-CD4 (RM4.5) or anti-CD8α (Ly-2) immunohistochemistry (1:50; BD-Pharmingen). Primary and biotinylated secondary antibodies were added and colour reaction was obtained by sequential incubation with avidin-peroxidase and 3-amino-9-ethylcarbazole, or alkaline phosphatase and Vector Blue AP III (SK-5300) (Vector Laboratories).
For evaluation of pancreatic cell division, sections were fixed for 15 min in 0.4% (wt/vol.) paraformaldehyde. Concomitantly to anti-insulin, rat anti-Ki-67 (1:100; Dako) and hamster anti-CD3-PE (BD-Pharmingen) were used. Detection was achieved by highly cross-adsorbed goat anti-guinea pig Alexa-647 and highly cross-adsorbed goat anti-rat Alexa-488 (Invitrogen, Carlsbad, CA, USA). The hamster anti-PE primary antibody was added after incubation with these detection antibodies to entirely eliminate cross-detection. Sections were mounted with Prolong Antifade reagent (Invitrogen) and images were captured with a confocal microscope (SP5; Leica, Wetzlar, Germany) equipped with a 20×/0.70 numerical aperture immersion objective (Leica). Renormalisation and background subtraction was performed using ImageJ software (http://rsbweb.nih.gov/ij/).
Insulitis scoring was performed according to the following criteria: (1) severe insulitis or score of 4 to 5, 50% or more of the islet area is infiltrated; (2) mild insulitis or score of 2 to 3, <50% of the islet area is infiltrated; (3) periinsulitis and no insulitis or score of 0 to 1, infiltration is restricted to the periphery of islets or absence of cell infiltration.
In vitro stimulation and adoptive transfer experiments
Lymphocytes from >35-week-old B:9-23/IFA-protected mice were pooled. Following a CD8-depletion step, cells were resuspended at 10 × 106/ml in 10% vol./vol. RPMI containing 50 U/ml rhIL-2 and 10 μg/ml B:9-23, and cultured for 3 days. Next, CD4+ cells were purified and: (1) stimulated for ICCS; or (2) further purified into CD4+CD25+ or CD4+CD25−, or into IFNγ+ and IFNγ− fractions. For ICCS, CD4+ cells were stimulated with plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (2.5 μg/ml) for 4 to 16 h with brefeldin A (10 μg/ml). CD4+CD25+ or CD4+CD25− cells were isolated with a kit (CD4+CD25+ Regulatory T cell Isolation Kit; Miltenyi Biotech, Auburn, CA, USA). For purification of IFNγ-producing cells, the total CD4+ cell fraction was stimulated in vitro with plate-bound anti-CD3 (4 μg/ml) and soluble anti-CD28 (2 μg/ml) for ∼14 h. IFNγ+ vs IFNγ− cells were collected using a mouse IFNγ secretion assay (Miltenyi Biotech) according to instructions. At least 1 × 106 total purified lymphocytes were adoptively transferred i.v. into 8- to 9-week-old NOD animals.
Anti-IL-10 (clone JES5-2A5) and anti-IFNγ (clone XMG1.2) were purchased from BD-Pharmingen. Mice (9 weeks old) were immunised with B:9-23/IFA and subsequently treated with 125 μg antibody i.p. This was done twice at 11 and twice 12 weeks of age, totalling four times (plus control [rat anti-mouse IgG1 isotype]). Mice receiving CD4+CD25+ T cells anti-IFNγ treatments started the next day (two times a week for 2 weeks).
Data are expressed as a mean ± SD. The statistical significance of the difference between means was determined using the two-tailed Student’s t test or the logrank test. Statistical tests were conducted with PRISM software (Graphpad, San Diego, CA, USA), with significance defined as *p < 0.05, **p < 0.01 and ***p < 0.001.
B:9-23/IFA immunisation s.c. can protect from advanced insulitis but not established diabetes
B:9-23/IFA treatment s.c. protects normoglycaemic mice from diabetes development
Blood glucose (mmol/l)
Mice treated (n)
Mice protected (n)
Mice protected (%)
Diabetic mice (n)
Mice fluctuatinga (n)
Prediabetic stage Ib
Prediabetic stage IIc
Prediabetic stage IIId
The above observations prompted us to test whether the same regimen could suppress ongoing diabetes. Blood glucose in NOD mice older than 10 weeks of age seems to reflect the degree of ongoing insulitis or alternatively the residual beta cell mass, i.e. mice with blood glucose >13.9 mmol/l are considered to have more insulitis and less beta cell mass than mice with blood glucose <13.9 mmol/l. Therefore, as indicated in Table 1, mice with blood glucose >10 mmol/l were divided into three groups and treated with B:9-23/IFA. Our results show that this type of intervention is moderately effective in mice at prediabetic stage III and with blood glucose no greater than 13.9 mmol/l. Mice from this group remained below 13.9 mmol/l during the total period of observation. When treated mice were plotted according to age, blood glucose at the time of treatment and progression to diabetes, an interesting trend emerged, namely that the earlier the disease manifestation and the higher the blood glucose, the more difficult it was to reverse hyperglycaemia (Fig. 1d).
Treatment with B:9-23/IFA s.c. reduces insulitis without enhancing beta cell proliferation
B:9-23 immunotherapy s.c. (1) preserved greater numbers of non-inflamed islets; (2) did not establish normal blood glucose during the first post-immunisation period in prediabetic NODs; and (3) restored normoglycaemia in some prediabetic stage III NOD mice. We therefore sought to address whether beta cell regeneration was involved. To test this, pancreatic sections were co-stained for insulin and Ki-67, and analysed histologically. Interestingly, as shown in Fig. 2c, few Ki-67/insulin double-positive beta cells could be detected, irrespective the treatment. Thus, we concluded that B:9-23/IFA immunisation did not affect beta cell replication, but instead modulated the autoreactive immune response, an observation that also explains why this regimen is not effective at more advanced stages of the disease, when greater beta cell mass is lost.
B:9-23/IFA therapy augments Tregs and results in loss of autoreactive CD8 cells
Since insulin is the initiating autoantigen in the NOD mouse, but other autoantigens such as GAD65 are recognised as the disease progresses , protection after B:9-23/IFA immunisation in older NOD mice was probably also mediated by bystander suppression mechanisms [25, 26, 27, 28]. To test this premise, cells from spleens of mice immunised with B:9-23/IFA were stained with the MHC class I tetramer NRP-V7, which identifies diabetogenic CD8+ T cells . As shown in Fig. 3i–j, a great reduction in NRP-V7+CD8+CD19− cells was seen in spleen, indicating that prevention of type 1 diabetes through B:9-23/IFA immunisation resulted in loss of NRPV-7 T cells.
B:9-23 immunisation increases protective B:9-23-specific IFNγ and IL-10 cytokine production
B:9-23/IFA-induced IFNγ supports the suppressive activity of CD4+CD25+ cells and shows immunomodulatory action
To further elucidate the immunoregulatory properties of IFNγ, CD4+IFNγ+ cells were adoptively transferred into new 8- to 9-week-old prediabetic hosts. It was found that this population of cells was able to delay, but not protect NOD recipients from diabetes development (Fig. 6c, d). On the other hand, IFNγ− cells failed to convey any protection, causing instead some acceleration of the disease. The fact that CD4+IFNγ+ cell transfer led to only transient protection from diabetes development could imply that (1) the number of cells transferred was limited (less than 1 × 106 cells/recipient), (2) pathogenic cells were also enriched, (3) too few regulatory cells were enriched or (4) the regulatory properties of those cells were lost with time.
Subcutaneous B:9-23/IFA treatment can effectively prevent diabetes when given at an early stage during disease development in NOD mice [14, 15]. Translation of this intervention to humans is in progress in early clinical trials , but it is not well known how the precise disease stage would affect therapeutic efficiency. In our study, B:9-23/IFA immunisation given at more advanced prediabetic stages significantly protected NOD mice from diabetes development, albeit to a less pronounced degree in mice with higher (non-physiological) blood glucose values (10–13.9 mmol/l). However, this intervention was not efficacious in mice that had established signs of diabetes and greater loss of beta cell mass, probably because B:9-23/IFA was not able to increase beta cell regeneration. Histological examination of mouse pancreases showed reduced lymphocytic infiltration, which was not associated with islet proliferation, suggesting that this intervention predominantly affected the autoaggressive effector response. This was due to the induction of Tregs that required IL-10 and IFNγ, but not IL-4 to be operational in vivo. Our findings are of particular interest for the ongoing B:9-23/IFA human trial, since induction of such Tregs could hopefully be tracked in human blood and serve as a correlate of successful immunisation and, in the future, maybe even as a secondary trial outcome.
It is likely that B:9-23/IFA immunisation mainly affected endogenous beta cell-specific, regulatory (protective) immune responses. This involved shifts in cytokine production and was associated with reduction of auto-reactive T cells of other specificities, such as IGRP-specific CD8 lymphocytes. The immune response against IGRP in NOD mice was also reduced in another study in which tolerance was achieved with intranasal proinsulin . Tregs in general play key roles in controlling many autoimmune processes. They can be identified by high levels of FOXP3 and IL-2Rα (CD25) and/or production of certain immunoregulatory cytokines [33, 34]. Their suppressive activity can be mediated by cell-to-cell contact or through cytokine production, with cytotoxic T lymphocyte antigen 4 (CTLA-4), IL-10 and TGF-beta-1 being the most important mediators of their function in vivo [35, 36, 37, 38]. In our study, augmented Tregs were found 2 to 3 weeks after immunisation (Fig. 3a–c). Whether these Tregs represent a de novo generated population from naive precursors or an expansion of the endogenous population is not known.
An increase in antigen-specific IFNγ, IL-4 and IL-10 production coincided with the time of Treg induction, since enhancement of B:9-23-specific IFNγ, IL-4 and IL-10 cytokine producing cells was seen 3 weeks after immunisation (Fig. 4). Neutralisation of IL-10 and IFNγ reversed protection, resulting in hyperglycaemia. Blocking IFNγ could also have diminished the action of autoaggressive lymphocytes, but in that case protection from disease after B:9-23/IFA treatment would still be seen. In order to address the contribution of IL-4 production to tolerance induction after B:9-23/IFA treatment, NOD/Il4 −/− mice were used. In these mice, progression to diabetes was still prevented after B:9-23/IFA treatment, suggesting that IL-4 production is not necessary for achieving tolerance. The contribution of TGF-beta-1 was not directly addressed in the present study, but its importance in mediating antigen-specific tolerance has been shown previously [39, 40, 41].
In order to further elucidate the immunomodulatory potential of the B:9-23-induced Tregs, cells from protected donors were isolated, expanded in vitro, separated into CD4+CD25+ and CD4+CD25− fractions and adoptively transferred into prediabetic hosts. The in vitro culture step seemed to be important for enhancing CD4+CD25+ regulatory properties, perhaps due to an increase in B:9-23-specific Treg qualities. Inclusion of cells from age-matched control mice was not possible since more than 90% of them developed diabetes, while cells from the remaining ones failed to grow under the same culture conditions. Additional analysis revealed a link between IFNγ/IL-10 production and Tregs, in that the CD4+CD25+ B:9-23-expanded cells were high producers of both cytokines, but not co-producers. In follow-up studies, IFNγ production was associated with protection from diabetes in CD4+CD25+ cell transfers. Recent evidence suggests that IFNγ can be important for protection from diabetes and other autoimmune diseases [42, 43, 44, 45, 46]. More recently in diabetes, IFNγ production was shown to suppress the pathogenic activity of Th17 cells . Upon B:9-23/IFA immunisation, we observed neither strong induction nor significant reduction of IL-17 production. While IL-17 levels remained constant, IFNγ production and the IFNγ:IL-17 ratio were increased. To further understand the contribution of IFNγ in B:9-23/IFA-induced tolerance, adoptive transfers with cells enriched in IFNγ production were done. A delay in disease progression was seen, suggesting that this population has at least transient regulatory properties and that other Tregs or other IL-10-producing cells are necessary for establishing prolonged tolerance.
In conclusion, IFNγ and IL-10 are important mediators of B:9-23/IFA-induced tolerance. Additional analyses will be required to understand the precise role of inflammatory cytokines such as IFNγ in Treg-mediated suppression of autoimmunity. To date, treatment with anti-CD3 antibody alone or in conjunction with nasal (pro)insulin therapy has been shown to reverse diabetes and to suppress disease recurrence . The capacity of InsB:9-23/IFA immunisation to reverse diabetes as a monotherapy or in conjunction with other drugs such as Abatacept (CTLA4-Ig) has not yet been tested. It is also possible that as antigens spread with disease progression, a combination of more than one autoantigen such as GAD65, GAD2 and IA-2 may be necessary to induce tolerance [13, 49]. Ideally, the use of more aggressive immunomodulatory regimens should be limited in time and dose, thus avoiding some of their negative side effects. This can be achieved by combining antigen-specific interventions with systemically acting agents.
We gratefully acknowledge support from Brehm coalition, NIH and Juvenile Diabetes Research Foundation funds. We would like to thank K. Coppieters, N. Amirian and M. McClure for technical help, and P. Colby for administrative assistance. G. Fousteri is an American Heart Association scholar award recipient.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
- 21.Fukushima K, Abiru N, Nagayama Y et al (2008) Combined insulin B:9-23 self-peptide and polyinosinic-polycytidylic acid accelerate insulitis but inhibit development of diabetes by increasing the proportion of CD4+Foxp3+ regulatory T cells in the islets in non-obese diabetic mice. Biochem Biophys Res Commun 367:719–724CrossRefPubMedGoogle Scholar
Open AccessThis is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License (https://creativecommons.org/licenses/by-nc/2.0), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.