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. 2015 Jan 2;179(2):245–255. doi: 10.1111/cei.12462

Reduced interferon-α production by dendritic cells in type 1 diabetes does not impair immunity to influenza virus

D Kreuzer 1, E Nikoopour 1, B C Y Au 1, O Krougly 1, E Lee-Chan 1, K L Summers 1, S M M Haeryfar 1, B Singh 1
PMCID: PMC4298402  PMID: 25286929

Abstract

The increased risk and persistence of infections in diabetic condition is probably associated with defects in the cellular immune responses. We have previously shown a decrease in the production of interferon (IFN)-α by dendritic cells (DCs) in diabetic subjects. The basal level of IFN-α in splenic plasmacytoid DCs (pDCs) is also lower in non-obese diabetic (NOD) mice compared to prediabetic mice. The objective of this study was to analyse the ability of diabetic mice to mobilize innate and CD8+ T cell-mediated immune response to influenza A virus (IAV) with the live influenza A/Puerto Rico/8/1934 H1N1 (PR8) strain or with its immunodominant CD8+ T cell epitopes. We found that following immunization with IAV, the level of IFN-α in diabetic mice was increased to the level in prediabetic mice. Immunization of NOD mice with the immunodominant IAV PR8 peptide induced clonal expansion of IFN-γ-producing CD8+ T cells similar to the response observed in prediabetic mice. Thus, diabetic and prediabetic NOD mice have a similar capacity for IFN-α and IFN-γ production by pDCs and CD8+ T cells, respectively. Therefore, the DC-related immune defect in diabetic NOD mice does not impair their capacity to develop an effective immune response to IAV. Our results suggest that reduced IFN-α production by diabetic human and mouse DCs is not an impediment to an effective immunity to IAV in type 1 diabetic subjects vaccinated with live attenuated influenza vaccine.

Keywords: cytotoxic T cells, dendritic cells, diabetes, vaccination

Introduction

Diabetic subjects have an increased susceptibility and severity to infections compared to non-diabetic subjects 1–3. This increased risk and the persistence of infections in diabetic subjects is due probably to a defect in innate immune responses in the diabetic condition. Recent studies have aimed to determine differences in the immune response mounted by diabetic and non-diabetic individuals 4,5. The non-obese diabetic (NOD) mouse is an animal model of human type 1 diabetes (T1D) that develops spontaneous autoimmune disease with a similar progression to that of human autoimmune T1D 6,7. We characterized endogenous dendritic cell (DC) levels in non-diabetic and diabetic human subjects and found no difference in the DC frequency, DC subset distribution and DC ability to activate T cells in both T1D and T2D subjects 5. However, the study revealed significantly reduced interferon (IFN)-α production by DCs from diabetic subjects 5. Other studies have also found a similar defect in DC function in diabetic NOD mice 8–13. Plasmacytoid dendritic cells (pDCs) are a relatively rare but important subset of DCs that are responsible mainly for type I IFN production 14,15. IFN-α is a very potent anti-viral cytokine produced by all nucleated cells in the early stages of the innate immune response due to recognition of a number of bacterial and viral antigens by Toll-like receptors (TLRs) and protein kinase R (PKR) 16. It is a major inducer of immature DC activation and as a consequence indirectly promotes cross-priming of CD8+ T cells against virally infected targets 16. IFN-α also modulates adaptive immunity by up-regulating co-stimulatory molecules on antigen-presenting cells (APCs) and by keeping CD8+ effector T cells alive and enhancing their proliferation 16.

In an attempt to investigate the impact of impaired DC function on innate and adaptive immunity in diabetic NOD mice, we used an influenza virus immunization model 17–19. We hypothesized that immunization with live influenza A virus (IAV) would induce IFN-α in diabetic mice. Secondly, we posited that immunization with live influenza virus and subsequent ex-vivo restimulation of cultured CD8+ T cells with an immunodominant peptide from IAV would induce the production of IFN-γ in CD8+ T cells in diabetic mice compared to prediabetic control mice.

We found that IFN-α production from pDCs of diabetic mice was increased to levels observed in the pDCs of prediabetic mice after influenza A immunization. We also found that immunodominant NP147-specific CD8+ T cells were equally responsive to their cognate peptide antigen in prediabetic and diabetic NOD mice. Thus, diabetic NOD mice are able to mount normal immune responses to IAV. Therefore, vaccination with attenuated IAV vaccines is likely to be a useful preventative measure for diabetic subjects against influenza infection despite reduced IFN-α production by DCs.

Materials and methods

Mice

NOD mice were bred and maintained in the pathogen-free barrier facility at the University of Western Ontario. The animal usage and care protocols followed the Canadian Council for Animal Care guidelines. Prediabetic female NOD mice 4–6 weeks of age were used for experiments requiring non-diabetic mice. Diabetic female NOD mice 20–26 weeks of age were used for experiments requiring diabetic mice and were confirmed to be diabetic by using Diastix glucose test strips.

Cell culture

Mouse spleens were extracted to prepare single-cell suspensions in 10% RPMI media. Red blood cells were lysed with ammonium–chloride–potassium (ACK) lysis buffer. Erythrocyte-depleted splenocytes were resuspended in complete RPMI media (Gibco) supplemented with 5 × 10−5 M 2-mercaptoethanol (ME), 1 unit/ml penicillin–streptomycin and 10% heat-inactivated fetal calf serum (FCS) (HyClone Laboratories, Logan, UT, USA). For induction of pDCs, splenocytes were stimulated in vitro for 3 days with 50 μg/ml Ep1B peptide. On the second day of culture, 10 μg/ml cytosine–phosphate–guanosine (CpG) (CpG ODN 1668; Sigma-Genosys, Oakville, ON, Canada) was added for the last 48 h.

Virus and peptides

The PR8 (Puerto Rico/8/34, H1N1) strains of IAV were propagated in 10-day-old embryonated chicken eggs and used as infectious allantoic fluid. Mice were injected intraperitoneally (i.p.) with ∼600 haemagglutinin units of either PR8 virus. All peptides used in this study were synthesized in our laboratory and purified by high performance liquid chromatography (HPLC), as before 20. Stock solution of peptides (1 mM) was prepared in dimethyl sulphoxide (DMSO) and stored at – 20°C. The Ep1.B (TQQIRLQAEIFQAR) peptide was dissolved in 10% glucose and passed through a 0·45 μm filter for sterilization. Peptides were used at a final concentration of 0·5 μM in intracellular cytokine staining protocol. For immunization, mice were injected i.p. with 50 μg of the indicated peptides in complete Freund's adjuvant (CFA) (Sigma, St Louis, MO, USA).

Antibodies

APC hamster anti-mouse CD11c (HL3), phycoerythrin (PE) rat anti-mouse CD45R/B220 (RA3-6B2), granzyme B and PE rat anti-mouse IFN-γ were from BD Biosciences (San Jose, CA, USA). APC-conjugated anti-mouse CD8α (Ly-2) was from eBioscience (San Diego, CA, USA). Fluorescein isothiocyanate (FITC)-conjugated rat monoclonal antibody (mAb) to mouse IFN-α was from PBL Biomedical Laboratories (Piscataway, NJ, USA). FITC rat immunoglobulin (Ig)G1 isotype control was from Southern Biotech (Birmingham, AL, USA).

IFN-α production by pDCs

IFN-α production in erythrocyte-depleted spleen cells was measured by staining with anti-IFN-α antibody. Brefeldin A (BFA) (Sigma, St Louis, MO, USA) was added to cells at a final concentration of 5 μg/ml to prevent secretion of newly synthesized IFN-α. Cells were incubated for 3 h at 37°C in humidified air with 5% CO2. Cells were washed and resuspended in 2% bovine serum albumin (BSA)/phosphate-buffered saline (PBS) (2% BSA). To block potential antibody FcR binding sites on cells, Fc-block (from 2·4 G2 hybridoma) was then added to cells for 20 min on ice and in the dark. Cell suspensions were stained with a 1:100 dilution of fluorochrome-conjugated anti-CD11c (0·2 mg/ml), mouse plasmacytoid DC antigen-1 (mPDCA) (0·2 mg/ml) and anti-B220 (0·2 mg/ml) for 45 min on ice and in the dark. Surfaced-stained cells were fixed for intracellular staining with 2% paraformaldehyde (PFA) for 10 min at room temperature (RT). Cells were then washed and 0·5% saponin in PBS (0·5% saponin) in 2% BSA was added to permeabilize cells. Next, FITC-labelled anti-IFN-α or FITC-labelled rat IgG1 (IFN-α isotype) was subsequently added. Cells were incubated on ice and in the dark for 45 min, washed and then resuspended in PBS for fluorescence activated cell sorter (FACS) analysis. Plasmacytoid DCs producing IFN-α were determined to be those with higher IFN-α intensity than the isotype control.

Characterization of anti-viral CD8+ T cells

To characterize peptide-specific CD8+ T cell responses, NOD mice were immunized i.p. either with live influenza A/Puerto Rico/8/1934 H1N1 (PR8) strain or with viral antigenic peptides in emulsified CFA. Cells were stained following a modification of previously described protocols 18,20. Erythrocyte-depleted spleen cells from immunized or control mice in RPMI-10 (2 × 105 cells/well) were added to a 96-well plate for peptide restimulation. Peptides NP39, HA518, HA462, NP218 and NP147 (Table 1) were then added at a final concentration of 0·5–1 μM in 200 μl media/well to appropriate wells. Plates were incubated for 2 h at 37°C in humidified air with 5% CO2. After 2 h, 5 μg/ml BFA was added to all wells. Plates were then incubated for a further 3 h. After blocking with Fc-block, cells were stained with anti-CD8 antibody. Plates were then incubated for 30 min on ice and in the dark. Cells were washed and fixed with 1% PFA for 20 min at RT in the dark. Antibodies for intracellular staining anti-IFN-γ PE and anti-granzyme B FITC were then diluted from stock concentrations at 1:200 using 0·5% saponin and added to the appropriate wells for at least 1 h on ice and in the dark. Stained cells were analysed using FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) and CellQuest Pro software. Further analysis was performed using FlowJo (version 8·6·1; Tree Star, Inc., Ashland, OR, USA) software.

Table 1.

In-vitro restimulation of spleen-derived memory CD8+ T cells from influenza A-immunized non-obese diabetic (NOD) mice with Kd-restricted peptides of influenza A nucleoprotein (NP) and haemagglutinin (HA)

Peptides Sequence
NP147–155 TYQRTRALV
HA518–526 IYSTVASSL
NP39–47 FYIQMCTEL
NP218–226 AYERMCNIL
HA462–470 LYEKVKSQL
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Statistical analysis

The data were analysed using the unpaired Student's t-test with Microsoft Excel software or Tukey's post-hoc analysis and a P-value of < ·05 was considered significant.

Results

Reduced level of pDCs and IFN-α in diabetic NOD mice

The baseline level of pDCs in prediabetic and diabetic NOD mice was investigated as a control to compare IFN-α production by pDCs in the same mouse model after influenza virus immunization. We found a reduced level of pDC numbers in diabetic mice compared with prediabetic mice as the basis for an immune defect in diabetic mice. Splenocytes were extracted from a diabetic and a prediabetic NOD mouse and stained for surface markers CD11c, mPDCA and intracellular IFN-α to characterize DC subsets by FACS. Diabetic mice displayed a lower percentage of mDCs (mPDCA-1− CD11chigh) and pDCs (mPDCA-1+ CD11clow) compared to prediabetic mice at basal conditions. The frequency of pDCs in spleens of prediabetic (n = 3) and diabetic (n = 3) NOD mice was 1·26 ± 0·14 and 0·65 ± 0·12, respectively, which was significantly different (P ≤ 0·05) (Fig. 1a). There was also a significant difference (P ≤ 0·05) for CD11c+IFN-α+ cells in prediabetic and diabetic mice (0·78 ± 0·07 and 0·35 ± 0·17, respectively) (Fig. 1b). As pDCs also express the B220 surface marker, we analysed IFN-α production in CD11c+B220+ cells. Similarly, there was a significant difference (P ≤ 0·05) in the frequency of CD11c+B220+IFN-α+ populations between prediabetic and diabetic NOD mice (0·91 ± 0·12 and 0·51 ± 0·19, respectively; data not shown). The reduced level of pDCs and IFN-α is related to the diabetic condition and not to the age of the mice, because the mice used in our study (6–25 weeks) are considered to be young to adult mice 21–24.

Figure 1.

Figure 1

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Reduced level of plasmacytoid dendritic cells (pDCs) in non-obese diabetic (NOD) mice compared to prediabetic NOD mice. (a) Frequency of DCs that express CD11c and mouse plasmacytoid dendritic cell antigen-1 (mPDCA) plasmacytoid DC markers in spleens of prediabetic and diabetic NOD mice. (b) Frequency of interferon (IFN)-α-producing pDCs in spleens of prediabetic and diabetic NOD mice. The figure is representative of three reproducible experiments. *P ≤ 0·05.

Reduced level of IFN-α production by pDCs from diabetic NOD mice are normalized after immunization

Basal level of IFN-α production by pDCs from prediabetic and diabetic NOD mice

Plasmacytoid DCs from prediabetic and diabetic NOD mice were examined for intracellular IFN-α levels (Fig. 2a).

Figure 2.

Figure 2

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Reduced level of interferon (IFN)-α production by plasmacytoid dendritic cells (pDCs) in non-obese diabetic (NOD) mice compared to prediabetic NOD mice is normalized by immunization with influenza A virus. (a) Percentage of IFN-α+ pDCs from prediabetic (top row) and diabetic (bottom row) NOD mice at basal levels (left column) compared to after 24 h of influenza A virus immunization (right column). (b) Frequency of splenic IFN-α+ CD11c after influenza A virus immunization. The figure is representative of four reproducible experiments. *P ≤ 0·05.

Immunization with influenza virus

In an attempt to correct the inherent innate immune defect, prediabetic and diabetic NOD mice were immunized i.p. with live influenza A virus. The effect of immunization on IFN-α production by pDCs was measured 24 h after inoculation. At baseline levels, diabetic pDCs produced 20% less IFN-α than prediabetic pDCs. However, IFN-α production by diabetic pDCs resembled IFN-α production levels by prediabetic pDCs after immunization (Fig. 2a).

The frequency of CD11c+IFN-α+ cells was determined in NOD mice at prediabetic and diabetic stages following immunization with live influenza virus (Fig. 2b). The frequencies of IFN-α+CD11c+ in spleen of prediabetic NOD mice before (n = 4) and after (n = 4) immunization with influenza virus were 0·97 ± 0·06% and 1·74 ± 0·4%, respectively (P ≤ 0·05). Diabetic NOD mice also showed an increase in frequency of splenic IFN-α+CD11c+ cells after influenza virus immunization: frequencies were 0·54 ± 0·12% before (n = 4) and 1 ± 0·5% after (n = 4) immunization with influenza virus (P ≤ 0·05). Despite this, the frequency of splenic IFN-α+CD11c+ cells in vaccinated diabetic mice was significantly lower than in vaccinated prediabetic mice (P ≤ 0·05). Overall, prediabetic and diabetic groups of NOD mice showed a 1·8- and 1·9-fold increase in frequency of IFN-α+CD11c+ cells, respectively. Thus, we conclude that vaccination with live influenza virus increases the numbers of splenic IFN-α+CD11c+ cells in diabetic and prediabetic mice, resulting in enhanced IFN-α production.

Immunization with influenza virus and ex-vivo restimulation with Ep1.B peptide and CpG

To understand more clearly the effect of immunization on IFN-α production by pDCs we used Ep1.B peptide and CpG as positive controls for the induction of pDCs. Ep1.B is a peptide fragment of apolipoprotein E that, when added to cell culture, enlarges the rare pDC population and increases the survival rate of these cells 20. CpG is a well-known ligand of intracellular endosomal TLR-9 that signals IFN-α transcription 16. Together, Ep1.B peptide and CpG have been shown to induce pDCs for proliferation, longevity and the production of IFN-α 25. Prediabetic and diabetic mice were immunized i.p. with live influenza A virus. After 24 h, splenocytes from immunized and non-immunized mice were incubated with Ep1.B peptide for 24 h and then with CpG for 48 h. Plasmacytoid DCs were analysed for intracellular IFN-α. Diabetic and prediabetic pDCs displayed a comparable ability to produce IFN-α after i.p. immunization with IAV and subsequent ex-vivo restimulation with Ep1.B and CpG (Fig. 3). Figure 3b shows that the frequency of CD11c+IFNα+ cells with or without live influenza virus immunization in the spleen of prediabetic and diabetic NOD mice (n = 4) was not significantly different.

Figure 3.

Figure 3

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Influenza A virus immunization does not alter interferon (IFN)-α production by splenic plasmacytoid dendritic cells (pDCs) in prediabetic and non-obese diabetic (NOD) mice by Ep1.B and cytosine–phosphate–guanosine (CpG) treatment of splenocytes. Splenocytes were stimulated in vitro for 3 days by 50 μg/ml Ep1B peptide. On the second day of culture, 10 μg/ml CpG was added. (a) Percentage of IFN-α+ pDCs after ex-vivo Ep1.B and CpG stimulation. (b) Percentage of IFN-α+CD11c pDCs after live influenza virus immunization and subsequent ex-vivo restimulation with Ep1.B and CpG. The figure is representative of three reproducible experiments.

NP147 peptide as the immunodominant epitope for restimulation of influenza-primed CD8+ T cells in NOD mice

We first demonstrated an immunodominant peptide hierarchy in the NOD mouse CD8+ T cell response to IAV. This permitted us to analyse cytokine production from an expanded population of CD8+ T cells and thus compare the adaptive immune response between diabetic and prediabetic mice. An influenza peptide immunodominance hierarchy described previously in the BALB/c mouse 17 was investigated in NOD mice. As BALB/c and NOD mice share the Kd MHC class I molecule they are likely to have same hierarchy for IAV. Splenocytes isolated from NOD mice (n = 3) immunized i.p. with live IAV were incubated with the IAV antigenic peptides NP39, HA518, HA462, NP218 and NP147. The capacity of each peptide to induce influenza-specific CD8+ T cell clonal expansion was examined. In NOD mice, NP147 had the strongest inductive effect on producing CD8+/IFN-γ+ effector T cells (Fig. 4). NP147 induced an approximately fourfold increase in the percentage of these cells compared to the no peptide-stimulation control.

Figure 4.

Figure 4

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NP147 peptide is the immunodominant epitope for restimulation of influenza-primed CD8+ T cells in non-obese diabetic (NOD) mice as measured by interferon (IFN)-γ production. Percentages of CD8+/IFN-α + T cells from the total spleen-derived CD8+ T cell population from influenza virus-immunized mice (n = 3) after ex-vivo restimulation with influenza A-derived peptides. From left to right, graphs represent restimulation with no peptide, NP39, HA518, HA462, NP218 and NP147, respectively.

Increased cytotoxic CD8+ T cell response in diabetic NOD mice after influenza virus or immunodominant NP147 peptide immunization and subsequent ex-vivo NP147 peptide restimulation

Immunization with influenza virus and subsequent ex-vivo NP147 peptide restimulation

After investigating the innate immune response of diabetic and prediabetic mice, we examined the influenza-specific adaptive immune response mediated by cytotoxic CD8+/IFN-γ+/granzyme B+ T cells for a more complete picture. To explore this response, splenocytes isolated from prediabetic (n = 3) and diabetic (n = 3) NOD mice were immunized i.p. with IAV and incubated with the immunodominant influenza viral peptide NP147. The peptide-specific T cell response was then compared between diabetic and prediabetic mice. Diabetic mice exhibited a greater percentage of CD8+ T cells after i.p. influenza virus immunization and ex-vivo NP147 restimulation (15·9%) compared to prediabetic mice (11·2%) (Fig. 5a). They also exhibited a greater IFN-γ response after immunization with influenza virus and subsequent restimulation with NP147 (4·24 ± 0·2%) compared to prediabetic mice (2·22 ± 0·3%) (Fig. 5b,c). The increased CD8+ T cell responses in diabetic NOD mice (Fig. 5c) are due probably to increased IFN-γ-producing CD4+ T cells in diabetic mice 26,27. Moreover, the increased IFN-α production also increased CD8+ T cell responses 28,29.

Figure 5.

Figure 5

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Increased cytotoxic CD8+ T cell response in non-obese diabetic (NOD) mice after immunization with influenza A virus and subsequent ex-vivo NP147 peptide restimulation. (a) Percentage of CD8+ T cells from live cells after intraperitoneal (i.p.) influenza A virus immunization and subsequent ex-vivo NP147 peptide restimulation. (b) Percentage of interferon (IFN)-γ+/granzyme B+/CD8+ T cells from the gated live cells after i.p. influenza A virus immunization and subsequent ex-vivo NP147 peptide restimulation. The top right gate represents IFN-γ+/granzyme B+/CD8+ percentage of total live gated cells. (c) Percentage of IFN-γ+ D8+ T cells in vaccinated prediabetic and diabetic NOD mice. The figure is representative of three reproducible experiments. *P ≤ 0·05.

NP147/CFA immunization and subsequent ex-vivo NP147 peptide restimulation

Because influenza virus immunization elicited an increased immune response in both the prediabetic and diabetic mice, we next examined whether the CD8+ T cell response to immunodominant viral peptides was also enhanced. Prediabetic and diabetic NOD mice were immunized with the immunodominant NP147 peptide of IAV or the HA462 control peptide, which was found previously to be at the bottom of our immunodominance hierarchy for clonal expansion of primed CD8+ T cells. The percentage of CD8+ T cells in diabetic mice (11%) was higher than CD8+ T cells in prediabetic mice (6%) (Fig. 6a). Restimulation with NP147 peptide expanded the influenza-specific CD8+ T cells to similar levels in both diabetic and prediabetic NOD mice. CD8+ T cells were not expanded in the control group of mice injected with HA462 control peptide (Fig. 6b,c).

Figure 6.

Figure 6

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Increased cytotoxic CD8+ T cell response in non-obese diabetic (NOD) mice after NP147 peptide/complete Freund's adjuvant (CFA) immunization. (a) Percentage of CD8+ T cells from live cells after immunization with NP147/CFA and subsequent ex-vivo NP147 peptide restimulation. (b) Percentage of interferon (IFN)-γ+/granzyme B+/CD8+ T cells from live cells after immunization with NP147 or HA462 peptides emulsified in CFA or with CFA alone and subsequent ex-vivo peptide restimulation. (c) Frequency of IFN-γ+/granzyme B+/CD8+ T cells from live cells after immunization with NP147 peptide emulsified in CFA and subsequent ex-vivo peptide restimulation. The figure is representative of three reproducible experiments.

Discussion

A study of patients with type 1 diabetes versus age-matched control non-diabetic individuals has shown a twofold increase in infection risk leading to hospitalization and even death 1. This has led to the analysis of DCs in diabetic patients. DCs play a pivotal role in the innate immune response via the production and secretion of the potent anti-viral cytokine IFN-α and also in linking the innate response to the adaptive response via antigen-presentation to naive CD8+ T cells. A previous study from our laboratory, conducted by Summers . 5, discovered a normal frequency of total DC and DC subsets in human diabetes as well as a normal phenotype of DC subsets. However, we discovered reduced IFN-α production by DCs in patients with T1D that may interfere with T cell-mediated immune responses leading to this observed increased risk and persistence of infections 5. In this study we investigated the innate and adaptive immune responses to IAV in prediabetic and diabetic NOD mice. We also evaluated IFN-α production by pDCs and the CD8+ T cell responses to IAV-derived antigenic peptides 17–19.

We initially confirmed that the baseline level of IFN-α production in splenic pDCs was lower in diabetic mice compared to prediabetic mice by analysing whole splenocytes. This suggests an altered function of pDCs in diabetic NOD mice similar to that observed in human diabetic patients 5. To investigate this perceived altered function further, we examined the ability of pDCs of diabetic mice to respond to influenza immunization and produce IFN-α compared to prediabetic mice. We immunized both groups of mice with live IAV and allowed 24 h for the optimization of the innate immune response and then analysed stained pDCs by FACS analysis 15. Peak levels of IFN-α production in pDCs of diabetic and prediabetic mice after immunization were similar. However, pDCs of diabetic mice exhibited an enhanced response, and IFN-α production in pDCs from diabetic mice was normalized to prediabetic pDC production of IFN-α from a lower baseline level. These differences in IFN-α levels were not due to the age of the mice used in the study, but related to the diabetic condition 8–13. Earlier studies in mice using influenza virus have shown that age-related differences are observed only when young and adult (6–16 weeks) mice are compared with old (more than 60 weeks) mice 26–29.

We also immunized both groups of mice in the same manner, but then stimulated pDCs specifically with Ep1.B peptide and CpG to expand the rare pDC population and production of IFN-α for a more defined analysis. Stained pDCs were analysed after restimulation. Levels of IFN-α production from pDCs of diabetic mice were induced to mildly higher levels compared to levels of production in prediabetic mice. The pDCs from diabetic mice also exhibited a similar IFN-α response when stimulated only with Ep1.B and CpG, exclusive of IAV immunization, compared to prediabetic pDCs. PDCs have been described previously to express TLR-7 and TLR-9 selectively to signal rapid secretion of immense amounts of IFN-α following viral stimulation 16.

We also investigated the ability of diabetic mice to develop an adaptive virus-specific CD8+ T cell response to influenza immunization compared to prediabetic mice, hypothesizing that immunization would increase IFN-α production that would contribute to increased CD8+ T cell responses 24,25. To probe this, we identified an immunodominant influenza viral peptide to induce clonal expansion of CD8+ T cells primed in vivo with live IAV that enabled us to analyse the CD8+ T cell response more accurately in the treatment groups. Thus, we employed a panel of influenza peptides, shown previously to form an immunodominance hierarchy in BALB/c mice, to stimulate influenza-specific CD8+ expansion 17. We determined the most immunodominant peptide by observing which peptide induced the greatest percentage of CD8+/IFN-γ+ and CD8+/granzyme B+ T cells in NOD mice. It was clear that the NP147 peptide induced the greatest percentage of cognate CD8+ T cell expansion of the peptides investigated. Thus, we concluded that NP147 is immunodominant among H2-Kd-restricted IAV peptides 17 in NOD mice. We found that NP147 peptide stimulation enhanced the number of CD8+ T cells in diabetic and prediabetic NOD mice following immunization with either live IAV or NP147 peptide/CFA. Type 1 IFN has been shown to increase CD8 T cells following viral infection 24. We concluded that diabetic NOD mice could overcome their inherent immune defect and mobilize an appropriate adaptive immune response to live influenza virus immunization. It is important, however, to note that these results are restricted to this i.p. model of immunization and may not correlate with the natural exposure to influenza virus. Thus, vaccination with IAV or its immunodominant peptides induces increased IFN-α levels in diabetic mice and results in stimulating innate and adaptive immune responses to IAV.

In conclusion, a correlation of this result to human diabetic subjects suggests that an attenuated influenza or influenza peptide vaccine would have a protective benefit to diabetic subjects and the annual influenza vaccine is a useful preventative measure against influenza-specific co-morbidity despite a reduced DC function.

Acknowledgments

This study was supported by grants from the Canadian Institutes of Health Research (CIHR). S. M. H. holds a Canada Research Chair in Viral Immunity and Pathogenesis.

Disclosure

The authors have no financial conflicts of interest.

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