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Keywords:

  • Autoimmunity;
  • CD8+ T cells;
  • Dendritic cells;
  • Immuneregulation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Type 1 diabetes is a T-cell-mediated autoimmune disease in which autoreactive CD8+ T cells destroy the insulin-producing pancreatic beta cells. Vitamin D3 and dexamethasone-modulated dendritic cells (Combi-DCs) loaded with islet antigens inducing islet-specific regulatory CD4+ T cells may offer a tissue-specific intervention therapy. The effect of Combi-DCs on CD8+ T cells, however, remains unknown. To investigate the interaction of CD8+ T cells with Combi-DCs presenting epitopes on HLA class I, naive, and memory CD8+ T cells were co-cultured with DCs and proliferation and function of peptide-specific T cells were analyzed. Antigen-loaded Combi-DCs were unable to prime naïve CD8+ T cells to proliferate, although a proportion of T cells converted to a memory phenotype. Moreover, expansion of CD8+ T cells that had been primed by mature monocyte-derived DCs (moDCs) was curtailed by Combi-DCs in co-cultures. Combi-DCs expanded memory T cells once, but CD8+ T-cell numbers collapsed by subsequent re-stimulation with Combi-DCs. Our data point that (re)activation of CD8+ T cells by antigen-pulsed Combi-DCs does not promote, but rather deteriorates, CD8+ T-cell immunity. Yet, Combi-DCs pulsed with CD8+ T-cell epitopes also act as targets of cytotoxicity, which is undesirable for survival of Combi-DCs infused into patients in therapeutic immune intervention strategies.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Type 1 diabetes (T1D) is a T-cell-mediated immune disease in which insulin-producing beta cells are specifically killed by CD8+ T cells [1]. There is no cure for T1D yet, but several clinical immunotherapeutic strategies are evaluated in the last few years [2-5]. The need for new immunomodulating therapies remains, in particular those that modify autoimmunity selectively, rather than suppressing the entire immune system [6, 7]. Dendritic cells (DCs) play a key role in directing the immune system toward immunity or tolerance [8, 9]. Tolerogenic DCs direct the immune system toward a regulatory response to presented antigens. A phase I trial using genetically modified DCs to express low co-stimulatory molecules (CD40, CD80, and CD86) supports a safe application of tolerogenic DCs for the treatment of T1D [10]. We are developing an intervention therapy for T1D with Combi-DCs, modulated by vitamin D3 and dexamethasone, which next to a low expression of co-stimulatory molecules induces other tolerogenic properties on DCs such as stable expression of inhibitory receptors and an anti-inflammatory cytokine production [11]. Furthermore, Combi-DCs are able to selectively induce apoptosis of autoreactive CD4+ T cells and convert naive CD4+ T cells into antigen-specific Treg cells [11, 12]. These Treg cells can, in turn, change monocyte-derived mature dendritic cells (moDCs) into DCs with regulatory capacity [13]. Therefore, Combi-DCs loaded with islet-specific CD4 T-cell epitopes are equipped with properties to induce islet-specific immunotolerance in vivo in patients with T1D.

Islet antigen-specific CD8+ T cells are predominantly infiltrating human islets of Langerhans in insulitis, as shown in postmortem analysis of recent-onset T1D patients [14, 15]. These cells are the direct effectors killing beta cells and it might be desirable to target them with tolerogenic DCs [15, 16]. Therefore, we analyzed the interaction between Combi-DCs loaded with HLA class I epitopes and naïve or memory CD8+ T cells.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Naive CD8+ T cells cannot be primed by Combi-DCs

To investigate the effects of Combi-DCs interacting with CD8+ T cells, we isolated naïve CD8+ T cells (CD8+CD45RA+CCR7+ CD27+CD28+) and co-cultured these with CMV-peptide pp65-loaded Combi-DCs or moDCs and autologous feeders. CMV-specific CD8+ T cells only proliferate in the presence of their epitope. Proliferation of tetramer (HLA-A*0201/pp65 peptide) positive (Tm+) T cells was analyzed by CFSE dilution (Fig. 1). Combi-DCs hardly induced proliferation of the CD8+Tm+ T cells, whereas cells stimulated with moDCs did proliferate within the first 4 days of co-culture. Antigen-specific CD8+ T cells did not expand when primed with Combi-DCs within 10 days, while the expansion rate of moDCs-primed CD8+ T cells was on average 130-fold (Fig. 1). Phenotype analysis showed that a small part (30.4% ±11.3) of the naïve CD8+ T cells primed by the Combi-DCs converted to the memory phenotype, even though they scarcely proliferate and expand (Table 1 and Supporting Information Fig.1). Cytokines produced in supernatant were analyzed by luminex assay. IL-2 and IL-10 were undetectable, while we noted a modest increase in IL-5, IL-13, and IFN-γ in supernatant of moDCs co-cultures (data not shown). The remaining CD45RA+RO T cells were CCR7+, confirming the naïve status of these cells in the Combi-DC co-cultures.

Table 1. Conversion of naïve CD8+ T cells into memory CD8+ T cells after stimulation with Combi-DCs and moDCs alone or combined
Stimulator ratio moDCs:Combi-DCsNaïve CD45RA+ CD8+Memory CD45RO+ CD8+
  1. a

    Data represent percentage of cells ± SEM (n = 2-3).

1:06.1 ± 2.3a93.9 ± 2.3
1:0.24.0 ± 2.496.0 ± 2.3
1:0.510.2 ± 2.589.9 ± 2.5
1:127.5 ± 5.572.5 ± 5.5
1:235.6 ± 8.264.4 ± 8.1
0:172.4 ± 10.230.4 ± 11.3
image

Figure 1. Combi-DCs cannot prime naive CD8+ T cells, but are able to expand enriched memory T cells. On day 0, naive CD8+ T cells or enriched memory CD8+ T cells were isolated and stimulated by CMV-peptide-loaded Combi-DCs or moDCs together with autologous feeders (DC:T-cell:feeder 1:10:20). Results for both naive CD8+ T cells (top) and memory CD8+ T cells (bottom) are shown. A portion of the isolated CD8+ T cells was labeled with CFSE to analyze the proliferation rate of tetramer-positive CFSE-labeled T cells at day 4. Dotted-line histograms show undivided cells, light gray histograms are moDCs-stimulated CD8+TmCMV+ T cells, dark histograms show CD8+TmCMV+ T cells stimulated by Combi-DCs. On day 10, all cells were analyzed for phenotype and expansion rate. Plots showing “Specificity” are one representative tetramer staining out of four independent experiments. The expansion rate was calculated as the number of CD8+Tm+ T cells on day 10 divided by the number of CD8+Tm+ cells on day 0 and is shown as mean + SEM of n = 4 samples pooled from four experiments performed. Difference in naïve expansion p = 0.0075; difference in memory expansion p = 0.067, with paired Student's t-test.

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Combi-DCs suppress priming of naïve CD8+ T cells by moDCs

Since Combi-DCs did not prime naïve CD8+ T cells, we investigated whether Combi-DCs are unable to stimulate T cells due to low expression of co-stimulatory molecules and low IL-12 production [11, 17], or whether Combi-DCs can actively interfere with priming of naïve CD8+ T cells. moDCs were co-cultured with naïve CD8+ T cells and autologous feeders (ratio 1:10:20) and their cell numbers stayed similar in all wells. Combi-DCs were added into the co-cultures with increasing cell numbers (Fig. 2). Combi-DCs impeded the priming of naïve CD8+ T cells in a dose-dependent fashion, preventing almost fully the expansion of CD8+ T cells when mixed with moDCs at a 1:2 ratio (moDCs:Combi-DCs) (Fig. 2). Increasing numbers of the stimulated naïve CD8+ T cells kept the naïve phenotype with increasing doses of Combi-DCs added to moDCs (Table 1). TNF or TGF-β produced by Combi-DCs [18, 19] did not contribute to prevention of priming since adding anti-TNF or anti-TGF-β to the co-culture did not reverse the impaired priming by Combi-DCs (data not shown). To analyze whether the inhibition by Combi-DCs involves antigen presentation, we added Combi-DCs without CMV-peptide at different ratios to the CD8+-moDCs co-cultures (Fig. 2). In this case, inhibition of CD8+ T-cell priming by moDCs did not occur, showing that the impairment of priming involves TCR-MHC-peptide complex binding. Second, the impaired priming is not caused by nonspecific steric hindrance of Combi-DCs, demonstrating that Combi-DCs actively impair priming of naïve CD8+ T cells.

image

Figure 2. Combi-DCs actively impede priming of naïve CD8+ T cells in an antigen-specific manner. CMV peptide-loaded moDCs were co-cultured with isolated naive CD8+ T cells and autologous feeders at a ratio of 1:10:20. (A) CMV peptide-pulsed Combi-DCs were added to the co-culture at different ratios ranging from 1:0.2 to 1:2 (moDC:Combi-DC). After 10 days, the percentage of Tm+ T cells was analyzed. Data shown are representative of three experiments performed. (B) Next to pulsed Combi-DCs (light gray bars), unpulsed Combi-DCs (black bars) were similarly cultured at different ratios with moDCs and naïve CD8+ T cells. Naïve CD8+ T cells were primed by peptide-loaded moDCs in the co-culture with different amounts of unpulsed Combi-DCs. Data are shown as mean + SEM of two to three independent experiments.

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Tolerogenic DCs purge antigen-specific memory CD8+ T cells after re-challenge

Similar to naïve CD8+ T cells, memory T cells were co-cultured with moDCs or Combi-DCs and autologous feeders. Within 4 days, both moDCs and Combi-DCs stimulated the proliferation of CD8+ memory T cells, although the Tm+ T cells stimulated with moDCs divided more than with Combi-DCs (proliferation index: 2.7 versus 1.9), leading to a threefold higher expansion of memory CD8+ T cells stimulated by moDCs than the expansion with Combi-DCs (Fig. 1). Nevertheless, the CMV-epitope-specific T cells stimulated by Combi-DCs had a similar phenotype to moDC-stimulated T cells and were able to kill their specific targets equally compared with T cell stimulated by moDCs (Supporting Information Fig. 2).

Memory CD8+ T cells were re-stimulated twice with either moDCs or Combi-DCs and analyzed (Fig. 3). The CMV-specific T cells were maintained in the cultures stimulated consecutively with moDCs, whereas Tm+ cells were diminished in the cultures stimulated twice with Combi-DCs (p = 0.026; Fig. 3). To test whether the reduction of the specific memory CD8+ T cells can be reversed by the second stimulation with moDCs, CMV-specific CD8+ T cells from the primary co-cultures with Combi-DCs were re-stimulated with moDCs (Fig. 3). The second stimulation with moDCs increased the percentage and absolute cell number of Tm+ memory CD8+ T cells compared with the cells stimulated twice with Combi-DCs, but the cells did not expand as well as cells stimulated twice with moDCs. To analyze the cell death during the second co-culture, we measured the percentage of Tm+ PI T cells. There was an increased percentage of dead Tm+ memory T cells in the cultures re-stimulated with Combi-DCs compared with the cultures stimulated with moDCs, regardless of the first stimulation (Fig. 3).

image

Figure 3. The second stimulation with Combi-DCs diminishes memory CD8+ T cells. Memory CD8+ T cells stimulated once were re-stimulated with moDCs or Combi-DCs for 10 days. (A) The percentage of Tm+ T cells stimulated twice with Combi-DCs compared with memory CD8+ T cells stimulated twice with moDCs is shown. The percentage of Tm+ CD8+ T cells in the cultures stimulated first with Combi-DCs and then with moDCs is also shown. Plots are representative of three independent experiments. (B) Expansion was calculated as cell number at day 20 divided by starting cell number at day 10. The numbers of Tm+ memory CD8+ T cells stimulated twice by Combi-DCs (Combi), stimulated twice by moDCs and stimulated first with Combi-DCs and subsequently with moDCs are shown. Symbols represent different donors; data are shown as mean ± SD of three experiments. Paired t-test: moDC/moDC versus Combi-DC/Combi-DC p = 0.026; moDCs/moDCs versus Combi-DCs/moDCs p = 0.10; Combi-DCs/moDCs versus Combi-DCs/Combi-DCs p = 0.10. (C) During the second co-culture, the percentage of dead Tm+ T cells was measured by staining with PI at different time points. Dead Tm+ T cells stimulated twice by Combi-DCs (black line) moDCs (dotted black line) and moDC/Combi-DCs (dotted gray line) are shown. Data are presented as mean ± SEM of two experiments performed in triplicate.

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Tolerogenic DCs loaded with CD8+ epitopes are targets of CTLs

Mature DCs have protective mechanisms due to killing by cytotoxic T-lymphocytes (CTLs) that include Serpin 9, an inhibitor of granzyme B [20], and Combi-DCs express less Serpin 9 than moDCs [21]. We tested whether CTLs kill Combi-DCs using a 51Chromium-release assay. Combi-DCs tended to resist CTLs killing better than moDCs (Fig. 4). Combi-DCs expressed HLA class I on average threefold lower than moDCs (MFI: 650 ± 45 versus 1983 ± 167; Fig. 4), which may contribute to the reduced killing of Combi-DCs compared with that of moDCs. Blockade of FASL or TNF did not abrogate killing of DCs, whereas inhibition of granule exocytosis with EGTA prevented killing of DCs by CTLs (Fig. 4). In conclusion, both moDCs and Combi-DCs were killed by CTLs by a perforin/granzyme B-mediated mechanism.

image

Figure 4. Combi-DCs are slightly more resistant to CTL killing than moDCs. DCs and B cells were labeled with 51Chromium and co-cultured with CTLs at different effector:target ratios for 4 h. (A) The percent of lysis of DCs and B cells by CTLs is shown as mean + SEM of n = 5 samples. p = 0.069 (paired Student's t-test). (B) Surface expression of HLA-ABC is shown on Combi-DCs (black line) and moDCs (gray line). Dotted line represents isotype control. Representative of three DC donors is shown. (C) To assess which pathway was used by the CTL clone to kill the DCs, we blocked Granzyme B/Perforin, TNF, and FAS pathways using a similar setup as in (A). Inhibitors were added to the assay-plate prior to the target cells. Killing of moDCs and Combi-DCs with (black circles, straight line) or without CMV peptide (black circles, dotted line) was assessed by percent lysis by CTLs alone or in the presence of Granzyme B/Perforin (empty circles), TNF (gray squares), and FASL (gray triangles) inhibitor. Data are shown as mean ± SEM of a representative experiment of three independent experiments, performed in triplicate.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

To assess whether Combi-DCs qualify to directly impair adaptive CD8+ T cells, in addition to their selective modulation of CD4+ T-cell immunity, we comprehensively studied naive and memory CD8+ T cells in co-culture with epitope-loaded Combi-DCs. Indeed, Combi-DCs impede priming of naïve CD8+ T cells, which is antigen dependent, but independent of TNF or TGF-β. Combi-DCs stimulate memory CD8+ T cells less vigorously than moDCs and, when co-cultured twice with Combi-DCs, memory CD8+ T cells are diminished during subsequent co-culture. Nevertheless, a drawback of loading of CD8+ T-cell epitopes onto Combi-DCs is that this renders Combi-DCs as target of CTLs. Taken together, our data suggest that the use of beta cell-derived HLA class I epitopes on Combi-DCs to intervene in CD8+ islet autoimmunity in T1D may be detrimental for autoreactive CD8+ T cells, but it would also reduce the survival and function of therapeutic Combi-DC products.

Impairment of priming naïve CD8+ T cells is a known action of Treg cells [22-24], but had not yet been described for tolerogenic DCs. Naïve CD8+ T cells require antigen recognition, co-stimulation, and activating cytokines (mostly IL-12) to become efficient effector T cells [17]. Combi-DCs express lower levels of co-stimulatory molecules and IL-12 [11] that may possibly provide incomplete stimulation to CD8+ T cells. Our data show that hampered stimulation as well as an active repression by Combi-DCs prevents priming of naive CD8+ T cells. Combi-DCs produce TNF and TGF-β [18, 19], but neither of these cytokines contributed to the obstructed priming of CD8+ T cells in our experiments. Since Combi-DCs interfere with priming only when they present the cognate antigen to the T cell, mechanisms related to TCR signaling rather than soluble factors may be responsible for this effect.

Combi-DCs can convert their tolerogenic properties to other cells, a mechanism referred to as infectious tolerance [13]. We investigated whether naïve CD8+ T cells co-cultured with Combi-DCs gain a phenotype or cytokine secretion profile related to Treg cells. The phenotype of CD8+ Treg cells is poorly defined and subject of debate. One report showed that human tolerogenic DCs can induce functional CD8+ Tregs expressing high levels of CTLA-4 and producing IL-10 [25]. In our experiments, functional tests were precluded due to low T-cell numbers after co-culture of naïve CD8+ T cells with Combi-DCs. The remaining few Tm+ CD8+ T cells did not express CTLA-4 (surface or intracellular), GITR (glucocorticoid-induced TNFR-related protein) and mTGF-β, while IL-10 was undetectable in the culture supernatant. Based on the phenotype and our finding that the majority of naïve CD8+ T cells stimulated with Combi-DCs retain a naïve phenotype, we conclude that tolerogenic DCs modulated by vitamin D3 and dexamethasone impair priming, rather than induce regulatory CD8+ T cells.

Combi-DCs initially expand memory CD8+ T cells, but CD8+ T cells collapse after the subsequent re-stimulation, leading to increased cell death in the second co-culture with Combi-DCs. Since CD8+ T cells initially stimulated with Combi-DCs do not expand, even when re-stimulated with moDCs, we envision that during the first stimulation Combi-DCs endorse memory CD8+ T cells with features underlying the subsequent ceased growth. The depletion of CD8+ T cells by the Combi-DCs could be the result of a combined action of the withdrawal of positive signals (co-stimulation and cytokines) or inhibition by secreted regulatory factors (such as IL-10 and TGF-β) that create a less favorable environment for cytotoxic T-cell survival and expansion [26, 27].

DCs have the ability to protect themselves from being killed by granzyme B-mediated mechanism [28]. Although Combi-DCs were less sensitive to killing than moDCs, possibly because of their lower HLA class I expression, 25–30% of peptide-pulsed Combi-DCs were killed by CTL within 4 h. In our view, using HLA class I peptide-loaded Combi-DCs for immunomodulatory cell therapy to exploit their inhibiting action on autoreactive CD8+ T cells does not appear to match the risk of these Combi-DCs being killed by the same effectors. Therefore, CD8+ T-cell epitopes appear less useful in Combi-DC therapy of autoimmune or autoinflammatory diseases in which memory T cells pre-exist and should be avoided to extend survival of Combi-DCs in therapeutic immune intervention strategies. Yet, in a prevention context where memory cells are not present, Combi-DCs pulsed with CD8+ T-cell epitopes may prove useful.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Generation of human DCs

The local ethical committee approval was received and informed consent of all participating subjects was obtained. The protocol of generating moDCs and Combi-DCs has been described [21]. In short, PBMCs from HLA-typed buffy coats obtained from healthy blood donors were isolated. Monocytes were isolated by positive selection using CD14-MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in RPMI-1640 medium with 8% FCS (Greiner Bio-One, Alphen aan den Rijn, Netherlands), recombinant human IL-4 (500 U/mL, Invitrogen, Breda, Netherlands) and recombinant human GM-CSF (800 U/mL, Invitrogen) for 6 days. Culture medium, including supplements, was refreshed on day 3. On day 6, the resulting immature DCs were activated by addition of 100 ng/mL LPS (Sigma-Aldrich Chemie, Zwijndrecht, Netherlands) for 2 days. To induce tolerogenic DCs, vitamin D3 (10−8 M, Sigma-Aldrich) was added on day 0 and refreshed on day 3 and dexamethasone (10−6 M, Sigma-Aldrich) was added on day 3.

CD8+ T-cell isolation, labeling, and cultures

Naïve CD8+ T cells were isolated from HLA-A*0201 PBLs using the naïve CD8+ T-cell isolation kit according to the supplier's protocol (Miltenyi Biotech). In short, naïve T cells were isolated by depletion of the nonnaïve T cells via magnetic separation followed by isolation of the CD8+ cells through positive magnetic selection. To obtain the enriched memory cells, CD8+ T cells were sorted using the negative isolation kit (Dynabeads® Untouched™ Human CD8+ T cells, Invitrogen), followed by the separation from naïve CD8+ T cells using the MACS isolation (Miltenyi Biotech). To measure proliferation rate, at least 1 × 106 of isolated CD8+ T cells were labeled with 1 μM CFSE [19]. Proliferation was analyzed after 4 days.

Isolated CD8+ T cells were cultured with autologous mature DCs (moDCs) or vitamin D3 and dexamethasone-modulated DCs (Combi-DCs) loaded with CMV-peptide (pp65, sequence NLVPMVATV; 1.5 μg/mL) at a 1:10 ratio in IMDM (Lonza, Verviers, Belgium) supplemented with L-glutamine (2 mM, Gibco), penicillin (100 U/mL, Gibco), streptomycin (100 μg/mL, Gibco), and 10% human serum in a round-bottom 96-well plate (Corning Costar). Irradiated autologous PBLs were added to the culture in a 1:2 CD8+ T-cell:PBL ratio to provide CD4+ T-cell help to the CD8+ T cells. One round of DC stimulation lasted 10 days. On day 4 of the stimulation round, medium was refreshed and IL-2 (10 U/mL), IL-7 (10 ng/mL), and IL-15 (5 ng/mL) (all from Peprotech, Rocky Hill, USA) were added to the culture. On day 7, the medium was refreshed again supplemented with IL-7 (10 ng/mL) and IL-15 (5 ng/mL). For the combined moDC/Combi-DC stimulation, moDCs with naïve CD8+ T cells and autologous PBL were cultured in a ratio 1:10:20, respectively. Combi-DCs were either loaded or not loaded with peptide and added into the culture at different ratios starting from 0.2:1 to 2:1 (Combi-DCs:moDCs). For the inhibition experiments, anti-TNF (10 μg/mL; Adalimumab, Humira®) or anti-TGF-β (20 ng/mL; clone TB21; IQ Products; Groningen, the Netherlands) was added to the co-culture at day 0 and CD8+ T cells were analyzed after 10 days. Cell death was measured by the percentage of PI (Milteny) positive, tetramer positive T cells in cell culture after 1, 5, and 10 days. Supernatant from co-cultures were harvested at day 10. Cytokine analysis was done with the Luminex 17-plex kit of Bio-Rad according to the manufacturer's protocol.

Phenotype analysis

After DC stimulation, the number of cells able to recognize the CMV-peptide was determined by FACS using a PE-labeled tetramer specific for the CMV-peptide (pp65, NLVPMVATV /HLA*0201 complex). After 10 days, the phenotype of the T cells was determined by staining with labeled antibodies: anti-CD8+ allophycocyanin (clone SK-1), anti-CD8+ PECy7 (clone SK-1), anti-CD45RA FITC (clone L48), anti-CD45RO APC (clone UCHL1), anti-CCR7 PeCy7 (clone 3D12), anti-CD25 allophycocyanin (clone M-A251), anti-CD27 FITC (clone M-T271), anti-CD28 FITC (clone CD28.2), anti-CD38 FITC (clone HB-7), CXCR3 allophycocyanin (1C6), and CD69 Pe-Cy5 (FN50). IgG1 FITC (clone X40), IgG1 PE (clone X40), IgG1 allophycocyanin (clone X40), IgG2a Pe-Cy7 (clone R35-95), anti-FITC HLA-ABC (clone W6/32). All from BD Pharmingen (San Diego, USA). FAS FITC (clone DX2) and PD-L1 allophycocyanin (MIH1) were from eBiosciences (San Diego, USA). The flow-cytometric stainings were analyzed on an FACS Calibur (Becton Dickinson, Breda, The Netherlands). Analyses were performed on Flow Jo 7.5 (Tree Star, Ashland, OR, USA).

Cytotoxicity assays

Target cells were labeled with 100 μCi of 51Cr and, when appropriate, pulsed with relevant peptide for 1 h at 37°C. Labeled cells were washed three times and mixed with the effector cells at various effector-to-target ratios and cultured at 37°C. Cytotoxicity was determined after 4 h by calculating the percent of specific lysis from counts of 51Cr released:

  • display math

The inhibitors used for the cytotoxicity assays were added at the start of the co-culture. Anti-TNF (Adalimumab, Humira®) at 10 μg/mL and anti-FAS Ligand (NOK1, eBiosciences) at 100 ng/mL. Granule exocytosis was inhibited by the calcium chelator EGTA (4 mM; Sigma-Aldrich). The CMV effector T-cell clone is a kind gift of Professor Frans Claas.

Statistical analysis

Data were compared by a two-tailed Student's t-test (paired or unpaired, as indicated in the figures) using the SPSS software package. Results were obtained from at least three independent experiments and presented as the average ± SEM, unless indicated otherwise.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

We thank Prof. Frans Claas for his useful comments and for kindly providing the CMV T-cell clone. FSK, AMJ, SL, and BOR were supported by a VICI grant of The Netherlands organization for scientific research (VICI, 918.86.611). JT and TN were supported by the European Union (NAIMIT, number 241447 in the FP7). BOR was supported by the Dutch Diabetes Research Foundation and the Juvenile Diabetes Research Foundation.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information
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Abbreviations
Combi-DC

vitamin D3 and dexamethasone-modulated dendritic cell

moDC

monocyte-derived mature dendritic cell

T1D

Type 1 diabetes

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

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eji2474-sup-0001-FigureS1.pdf335K

Figure S1. Gating strategy for the analysis of CD8+ t-cells.

Figure S2. Memory CD8+ T cells expanded by Combi-DCs or moDCs have a similar phenotype and function.

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