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Keywords:

  • DC;
  • TCR transgenic mice;
  • Transplantation tolerance

Abstract

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

Pharmacological modulation is known to temper the immune capacity of DC, enhancing the notion that modulated Ag-bearing DC might be used therapeutically to induce tolerance. We have investigated phenotypic features shared by such DC, and queried their potential to tolerize in different settings. Immature, IL-10, TGF-β and 1α,25-dihydroxyvitamin D3-modulated BMDC all induced tolerance to male skin in female TCR transgenic A1.RAG mice, and the modulated DC also tolerized after exposure to the TLR4-ligand LPS. Transcript profiling revealed that this was achieved despite retaining much of the normal LPS-maturation response. No shared tolerance-associated transcripts could be identified. Equivalent BMDC could not tolerize in Marilyn TCR-transgenic mice. Simultaneous presentation of both A1.RAG and Marilyn peptide-Ag (Dby-H2Ek and Dby-H2Ab) on immature (C57BL/6JxCBA/Ca) F1 BMDC also only achieved tolerance in A1.RAG mice. Both strains registered Ag, but Foxp3+ Treg were only induced in A1.RAG mice. In contrast, Marilyn T cells showed greater proliferation and an inflammatory bias, in response to Ag presented by immature F1 BMDC in vitro. In summary, while pharmacological agents can skew DC to reinforce their immature tolerogenic phenotype, the outcome of presentation is ultimately an integrated response including T-cell-intrinsic components that can over-ride for immune activation.


Introduction

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

Despite enthusiasm to exploit tolerogenic DC clinically 1–3, what constitutes a tolerogenic DC in vivo, and how it achieves tolerance, is still poorly understood. Immunological outcome was thought to be related directly to the maturation status of DC, with immature DC presenting for tolerance 4–6; however, this simple view rapidly failed to account for accumulating experimental observations 7, 8. The functional diversity of DC has more recently been explained by the integration of multiple factors within a particular anatomical location causing the differentiation of appropriately tuned “effector” DC 9, 10. Although key co-inhibitory receptors, such as ILT3/4, PIR-B, PD-L1 and ICOSL, and immunoregulatory molecules, such as IDO and histone deacetylase HDAC11, have been variously implicated in driving tolerance 11–16, it is still not clear to what degree such molecules are universal players.

A concern using immature DC in vivo is that they might become activated, particularly within the context of inflammation. If DC are to be used therapeutically, then the tolerant phenotype must be robust and stable. Numerous agents have been used to manipulate DC in vitro to generate maturation-resistant cells for adoptive cell therapy 2, 9; however, attempts to induce tolerance to allografts in conventional models have generally failed unless combined with other immunosuppressive agents 1, necessitating a move to reductionist models. We previously showed that we could induce tolerance to male skin grafts in female TCR transgenic A1.RAG mice by transferring either “immature” BMDC or BMDC modulated using 1α, 25-dihydroxyvitamin D3, (BMDC-VD3), but only the modulated cells retained their capacity to induce tolerance after maturation with the TLR4-ligand LPS 17. The tolerance induced was long-lasting, dominant and associated with de novo generation of peripheral Foxp3+ T cells. Here, we have established a series of tolerogenic BMDC modulated with different agents and have tried, but failed, to identify a common “tolerant” molecular signature. An equivalent series of modulated BMDC could not tolerize in a different male-specific TCR transgenic strain, Marilyn. Using F1 BMDC, able to present Ag to both transgenic strains, we demonstrate that tolerance is not a dominant DC characteristic, but can be overridden by T-cell-intrinsic factors.

Results

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

DC exposed to different biological modifiers induce tolerance in A1.RAG mice

All of the T cells in naïve A1.RAG TCR transgenic mice are CD4+ and recognise a defined peptide of the male-specific Ag Dby, presented in the context of H2Ek18. Having demonstrated that BMDC-VD3 can induce tolerance in A1.RAG mice 17, we now asked if BMDC influenced by the immunomodulatory cytokines IL-10 (BMDC-IL10) and TGF-β (BMDC-TGF) could do the same. Both of the cytokine-modulated populations induced long-term transplantation tolerance even after LPS maturation (Fig. 1A and B), and de novo expression of Foxp3 was detected in CD25+ splenic T cells from tolerized hosts (Fig. 1C). Foxp3+CD4+CD25+ T cells were also detected in spleens from mice pre-treated with mature immunogenic BMDC+LPS, demonstrating that induction of Foxp3 per se does not preclude rejection, but rather in this case regulation was insufficient to prevent loss of the graft. Splenic T cells from tolerized mice were profoundly hypo-responsive to Ag in vitro, with only low levels of IL-2 detected in culture supernatants (Fig. 1D). Proliferation of T cells from rejection-competent mice pretreated with BMDC+LPS was also reduced, although the associated level of IL-2 production was comparable to non-tolerant controls.

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Figure 1. “Immature” and cytokine-modulated BMDC induce male-specific tolerance in A1.RAG mice with de novo induction of T cells with a regulatory phenotype. (A) Female A1.RAG mice received 2×106 of the indicated male CBA BMDC and were grafted 28 days later with male CBA.RAG skin. Grafts were monitored over a period of 100 days. Syngeneic female grafts were all accepted indefinitely (data not shown). (B) Mice grafted for > 40 days were re-grafted with a second male CBA.RAG skin. (Analogous data for BMDC-IL10±LPS are not shown.) (C) Splenocytes stained for CD3, CD4, CD25 and intracellular Foxp3 were analyzed by flow cytometry gated on CD3+CD4+ cells. Data shown are representative of two independent experiments performed using splenocytes obtained 40 and 100 days post-grafting (day 40 data are shown). (D) Nylon-wool purified splenic T cells isolated from grafted (40 days post-grafting) and from naïve untreated control mice were assessed by recall assay. Spleens from two mice were pooled for each sample. Similar data were obtained at 40, 80 and 100 days post grafting using total splenocytes. Proliferation is indicated as the mean incorporation of 3H-TdR±SEM for triplicate co-cultures. Levels of IL-2 in recall co-culture supernatants containing 10 nM of Dby peptide were assessed by ELISA.

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Searching for molecular patterns that correlate with DC tolerogenicity

A multiplex bead-based assay was used to assess secretion of soluble mediators by the different BMDC populations, and attempts were made to correlate this with their ability to induce tolerance in vivo (Supporting Information Fig. 1). The majority of the 27 mediators assayed were not detected in the absence of LPS regardless of the modulation regime. None were exclusively detected in, nor selectively absent from, all of the tolerance-prone DC supernatants. Modifier-specific effects on the degree of LPS responsiveness were observed, with IL-10-modulation tending to yield the least, and TGF- and VD3-modulation equivalent, or even variously enhanced, levels compared with non-modulated BMDC. In general, while relative trends were retained, absolute quantities increased in the presence of cognate T cells. No mediators were increased in all of the co-cultures containing tolerance-prone DC compared with those containing BMDC+LPS; however, levels of IL-2, IL-12p40, GM-CSF and RANTES were reduced (Supporting Information Fig. 1B).

We also searched for transcription-based signatures for tolerance. Candidate genes were initially identified using low-stringency statistical pattern searching within global serial analysis of gene expression (SAGE) and microarray data sets, and the relative expression of these, and other genes of interest, were determined in the different BMDC populations using quantitative PCR (qPCR) (Supporting Information Fig. 2A). The majority of the ∼150 transcripts analyzed by qPCR fell into two categories: equivalent in all populations, or increased in response to LPS regardless of modulation, and it was evident that LPS-matured modulated BMDC were able to achieve tolerance in vivo despite retaining much of the LPS-induced transcriptional response seen in non-tolerogenic BMDC+LPS. By statistical criteria, none of the transcripts analyzed were significantly increased, nor decreased in all of the tolerogenic BMDC relative to non-tolerogenic BMDC+LPS (Supporting Information Fig. 2B).

Tolerant DC did share similarities in surface phenotype. As for BMDC-VD3 17, surface expression of co-stimulatory molecules CD40 and CD86 was indistinguishable between the cytokine-modulated and untreated immature BMDC, with levels of MHC class II slightly down-modulated (Supporting Information Fig. 3). In contrast to untreated BMDC, all of the modulated populations were impaired to some degree in their ability to upregulate surface expression of these molecules in response to LPS, and this impairment was paralleled by a reduced capacity to stimulate Ag-specific proliferation of naïve T cells in vitro. The E3 ubiquitin ligases MARCH1 and MARCH8 are known to be involved in regulating MHC class II and CD86, with maturation-induced loss of ubiquitination promoting cell surface expression 19–21. Pronounced changes previously reported in the level of MARCH1 mRNA in human monocyte-derived DC, 20-fold on maturation and 40-fold in response to IL-10 22, 23, were not evident in our data (Supporting Information Fig. 2A). A very slight increase in MARCH1 transcription was observed in response to LPS in the modulated DC. We considered the possibility that alternative splicing could be altering the stability and function of MARCH1 by incorporating different N-termini 24; however, transcript variant 2 (NM_001166375.1) was the dominant form (∼1000-fold excess over other 5′ variants, NM_001166372.1 and NM_175188.4) in all of the BMDC (qPCR data not shown). MARCH8 was unchanged across all of the BMDC populations regardless of maturation.

Modulated BMDC induce tolerance in one, but not another male-specific TCR transgenic mouse

Having established that pharmacologically-modulated DC could tolerize in CBA-derived, A1.RAG mice, we asked whether they could also do so in B6-derived, TCR transgenic Marilyn mice. As in A1.RAG mice, all of the naïve T cells in Marilyn mice are CD4+Fop3 and they all recognise a specific peptide of the male Ag Dby, which in this case is presented by H2Ab25. Like CBA BMDC, modulated B6 BMDC were all impaired in their ability to up regulate surface levels of MHC class II and co-stimulatory molecules, and although B6 immature BMDC induced Ag-specific T-cell proliferation comparable to B6 LPS-matured BMDC, the inductive capacity of the modulated DC was once again reduced, regardless of exposure to LPS (Supporting Information Fig. 3B). Despite their similarities with tolerant CBA-modulated BMDC, B6 male BMDC, whether immature or modulated (BMDC-VD3 or BMDC-IL10), administered at doses ranging from 0.5×106 to 10×106 cells, all failed to promote acceptance of male skin grafts by female Marilyn mice (Fig. 2A). This failure to induce tolerance did not reflect an inherent inability of Marilyn mice to be tolerized to Dby, as tolerance was readily achieved using a single dose of non-ablative anti-CD4 Ab (Fig. 2B). Foxp3 mRNA was detected within the spleens and grafts of the anti-CD4 tolerized Marilyn mice, as previously described for A1.RAG mice 26 (Fig. 2C).

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Figure 2. Immature and modulated BMDC fail to induce tolerance in Marilyn mice. (A) Female Marilyn mice were injected with PBS, or with titrated numbers of immature or modulated male B6 BMDC and grafted 28 days later using male B6.RAG skin. Two mice receiving 10×106 immature BMDC did hold their grafts to > 100 days, but only after a prolonged rejection crisis. For clarity, the data are plotted across two graphs with the same control PBS mice included on both graphs. (B) A single dose of non-depleting anti-CD4 or isotype control Ab was given to female Marilyn mice at the time of grafting with male B6.RAG skin (day 0). Control male and female grafts were applied in the absence of Ab. Second male B6.RAG challenge grafts were applied after 100 days. The same mouse in the anti-CD4 group rejected both grafts. Syngeneic female grafts and re-grafts were all accepted indefinitely (data not shown). (C) At 80 days post re-grafting, Foxp3 mRNA levels were compared in splenocytes and in surviving skin grafts (primary, 1st; regraft, 2nd). Skins from control mice grafted with female skin and archived skins from anti-CD4-treated A1.RAG mice were included. Foxp3 mRNA levels are shown normalized to CD3γ and relative to levels in naive B6 spleen (indicated by the dotted line). Error bars indicate ±SD of the geometric mean relative quantitation for biological replicates (splenocytes: anti-CD4, n=5; female graft, n=2. Grafts: anti-CD4 1st, n=5; anti-CD4 2nd, n=4; female 1st and 2nd, both n=2; tolerant A1.RAG n=5.)

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Immature (B6×CBA) F1 BMDC induce tolerance in A1.RAG, but not Marilyn mice

To account for any differential effects contributed by the strain background of the BMDC, we compared the outcome of presenting both strain-specific peptide-Ag, Dby-H2Ek and Dby-H2Ab, on the same (B6×CBA)-derived F1 BMDC. Using 2×106 immature male F1 BMDC, almost all of the treated A1.RAG mice (14/15) accepted strain-matched male grafts long-term (>80 days), while all of the Marilyn mice (9/9) once again rapidly rejected theirs (mean survival time, 13 days) (Fig. 3A). Co-expression of similar surface levels of H2Ek and H2Ab was confirmed by surface staining using a combination of shared and haplotype-specific anti-class II antibodies (Fig. 3B), and T cells from both mouse strains engaged Ag presented on F1 BMDC, as evidenced by increased levels of the early activation marker CD69 on splenic T cells monitored 23 h after injection of immature male (A1.RAG 9.5±0.1% and Marilyn 35±4%), but not female (A1.RAG 1.6±0.3% and Marilyn 1.3±0.4%) F1 BMDC (Fig. 3C). Foxp3 was only induced in the tolerizable A1.RAG strain (Fig. 3D). These contrasting immunological outcomes in response to the same F1 BMDC clearly demonstrate that the status of the presenting DC alone is insufficient to guarantee tolerance.

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Figure 3. Presentation by the same F1 BMDC induces tolerance in A1.RAG, but not Marilyn mice. (A) Female A1.RAG and Marilyn mice received 2×106 immature F1 male BMDC or PBS alone 28 days prior to application of strain-matched male skin grafts. Grafts surviving in the PBS controls were typical of chronically rejecting grafts; contracted, hairless, white and often shiny. (B) Immature and LPS-matured B6, CBA and F1 BMDC were stained in parallel using antibodies specific for H2Ab (presents to Marilyn T cells), H2Ek (presents to A1.RAG T cells) and H2Ab+H2Ek (2G9). Plots represent data from three experiments gated on live cells using forward and side scatter. Typically, ∼70% of each population were CD11c+. The maturation shift to bright MHC class II expression is equivalent, but more complete on CBA BMDC. The very small degree of scatter from the 45° line in the F1 BMDC dot plots suggests that H2Ab and H2Ek are accurately co-expressed. Similar staining observed using allele-specific antibodies (shown in the dot plots) and 2G9, staining both alleles, (shown in the histogram), strongly suggests that the co-expression is both qualitative and quantitative. (C) A1.RAG and Marilyn splenocytes isolated 23 h post-injection of 2×106 male or female F1 BMDC were stained for CD3, CD4 and CD69 and analyzed by flow cytometry. Cells were gated using forward and side scatter, and then on CD3+CD4+ cells. Each plot is representative of three mice. (D) Splenocytes isolated from experimental and PBS control mice 75 days after grafting were stained for CD3, CD4 and intracellular Foxp3 and analyzed by flow cytometry. Live cells were gated using forward and side scatter. Percent CD3+CD4+ cells staining for Foxp3 for individual mice are shown, with the group mean indicated by a horizontal bar.

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Marilyn T cells are more responsive to Ag presented on immature F1 BMDC

Comparing the responder T cells, we found that TCR expression was lower on A1.RAG compared with Marilyn splenic T cells (Fig. 4A). Proliferation was similar in response to anti-CD3 stimulation, although the A1.RAG response did plateau at a lower level (Fig. 4B), and Foxp3 was induced equivalently in vitro when stimulated with anti-CD3/CD28 beads plus 2 ng/mL TGF-β, (∼35–40% by FACS, data not shown). No direct measure of TCR affinity for the different peptide-MHC complexes is available; however, when stimulating with peptide-pulsed female F1 BMDC, approximately tenfold less Marilyn-specific peptide was required to induce equivalent T-cell proliferation compared with the A1.RAG peptide (Fig. 4C). Marilyn T cells also showed a heightened proliferative response to male F1 BMDC in vitro (Fig. 4E). Accumulation of IL-6, IL-17, IFN-γ and MIG was also increased in the supernatants of Marilyn T cells co-cultured with immature male F1 BMDC (Fig. 4F). Only IL-17 remained differential in the presence of stronger stimulation provided by LPS-matured F1 BMDC, which did not tolerize in either mouse strain. Both the responsiveness and the pro-inflammatory bias of the Marilyn interaction with F1 BMDC could favor rejection in vivo.

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Figure 4. Marilyn T cells display heightened responses to Ag presented on F1 BMDC. (A) Splenocytes stained for CD3 and CD4 were analyzed by flow cytometry gated on forward and side scatter. Quad gates were set using isotype control antibodies (not shown). Levels of CD3 expression in both CBA and B6 were comparable with Marilyn (data not shown). Data are representative of four experiments. (B) Female A1.RAG or Marilyn T cells were plated across a gradient of immobilized anti-CD3 Ab in the presence of 1 μg/mL of soluble anti-CD28 Ab. Proliferation is indicated as the mean incorporation of 3H-TdR ± SD for triplicate co-cultures. Data are representative of two independent experiments. (C) Female A1.RAG or Marilyn splenic T cells were co-cultured at a ratio of 15:1 with either female F1 BMDC ± LPS pulsed with the appropriate Dby peptide at the indicated concentrations, or alternatively with male F1 BMDC ± LPS (male). Data are representative of two independent experiments. (D) Female A1.RAG or Marilyn splenic T cells were co-cultured in vitro with F1 BMDC ± LPS. Cultures were harvested over a period of 40 h and stained for CD3, CD4 and CD69. Percent CD3+CD4+ T cells expressing CD69 was determined by FACS and is shown graphically. The time course was performed once, with the last time point repeated. (E) Female A1.RAG or Marilyn splenic T cells (2.5×104per well) were co-cultured in vitro with a titration of male F1 BMDC±LPS. Data are representative of three independent experiments. (F) Male F1 BMDC±LPS were replated on day 7 in the presence of female A1.RAG (white bars) or Marilyn (grey bars) splenic T cells and supernatants analyzed after 48 h using a 25-component multiplex bead-based assay. Cells plated alone and media incubated in the absence of cells were also analyzed (black bars). IL-6, IL-17, IFN-γ and MIG preferentially accumulated when immature F1 BMDC were co-cultured with Marilyn T cells. The y-intercept indicates assay detection limits. Error bars indicate±SD from the mean for three independently derived sets of supernatants.

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Discussion

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

Numerous studies linking tolerance to DC maturation status have emphasized the importance of incomplete signaling to T cells 4–8, while others have highlighted dominant interactions delivering negative or co-inhibitory signals as critical 11–14, 16. More complete explanations advocate some balance of the two, with micro-environmental context of antigenic encounter, and the nature of the Ag itself proposed as key determinants in establishing this balance of signaling 9, 10. We addressed the degree to which “universal” mechanisms are invoked by tolerogenic DC by comparing a set of male CBA-derived BMDC including DC modulated by VD3, IL-10 or TGF-β. Each of these DC populations induced tolerance to a male minor Ag in female A1.RAG TCR transgenic mice, and unlike immature BMDC, retained their tolerizing capacity even after TLR4 stimulation using LPS.

Despite extensive mRNA profiling, we could not identify any transcripts whose expression consistently marked all of the seven tolerant BMDC populations, or alternatively only marked non-tolerant BMDC+LPS. We did, however, notice a general trend for LPS-induced expression of various amino-acid catabolic enzymes, which could represent components of a redundant mechanism for mediating tolerance through depletion of essential amino acids 27. The modulated BMDC populations were clearly not refractory to LPS stimulation, but instead retained much of the normal LPS-induced transcription response. While some maturation of adoptively transferred tolerogenic DC is required to promote migration to the lymph nodes for Ag presentation to T cells 28, transcripts increased in response to LPS in this study encoded both classic pro-inflammatory agents, such as IL-1 and NOS2, and also well-characterized inhibitory molecules, such as IL-10, Indo and CD274 (PD-L1) 12, 16, 29 (Supporting Information Fig. 2A). Similarly, both pro- and anti-inflammatory soluble mediators were secreted by all of the LPS-stimulated BMDC, albeit at lower levels in the IL-10-modulated DC (Supporting Information Fig. 1). It is evident from these data that DC-mediated decisions for both tolerance and immunity are achieved within a context of multiple pro- and anti-inflammatory mediators.

Tolerogenic CBA BMDC all exhibited low surface levels of MHC class II and co-stimulatory molecules, but this was also a feature of non-tolerizing B6-derived BMDC (Supporting Information Fig. 1A). Ubiquitination of the cytoplasmic tails of MHC class II and CD86 by MARCH1 and MARCH8 is known to regulate their surface expression 19–21. Transcription of MARCH1 and MARCH8 was, however, essentially unchanged in all of the populations (B6 data not shown). This contrasts with previous studies using human monocyte-derived DC where downregulation of MARCH1 followed maturation, and a pronounced increase in MARCH1 was seen in response to IL-10 22, 23.

To eliminate genetic differences between the two mouse strains that might have determined the different tolerance inducing capacity of their DC, we compared the tolerogenicity of male immature (B6×CBA) F1 BMDC in both strains. Tolerance was again only achieved in A1.RAG mice, clearly indicating that the functional status of the DC alone is insufficient for tolerance. Although the A1.RAG and Marilyn TCR recognise peptides derived from the same endogenous male protein, Dby, they are presented on different MHC class II molecules, to different transgenic TCR expressed on T cells from different strain backgrounds. Both the dose and affinity of peptide-MHC complexes are known to affect the outcome of T-cell interactions 30, 31. Equivalent co-expression of surface H2Ek (presents to A1.RAG) and H2Ab (presents to Marilyn) by F1 BMDC implied that they have the capacity to present at an equivalent Ag dose to both mouse strains.

Using RAG-sufficient mice it has been suggested that A1 CD4+ T cells may be more susceptible to in vivo regulation than those from Marilyn mice, although it was not clear why this should be so 32. Marilyn mice are not incapable of tolerance as they were readily tolerized using non-depleting anti-CD4 Ab. Failure to tolerize was also not due to aberrant delivery of Ag, as T cells from both strains registered Ag encounter. In vitro, both strains demonstrated an equivalent capacity to induce Foxp3 in response to maximal TCR cross-linking in the presence of TGF-β (data not shown). TCR levels on Marilyn mice were higher than those on A1.RAG, and Marilyn T cells were generally more responsive to F1 BMDC±LPS. Under conditions of sub-optimal Ag presentation by F1 BMDC Marilyn T cells also appeared inflammatory-prone. The heightened responsiveness was not due to pre-existing memory T cells within the splenocytes of Marilyn mice (CD44 staining, data not shown), suggesting that the difference between the two strains reflect T-cell-intrinsic factors such as the avidity of their respective TCR/peptide:MHC interactions, and ability to evoke inflammatory cytokine production.

We have clearly shown that although DC can be skewed to favor presentation for tolerance, tolerance is not then an obligatory outcome in these “reductionist” TCR transgenic mice. Rather, the outcome represents an integration of combined signals occurring between the DC and the interacting T cell. Within a “polyclonal” mouse, these interactions are almost certain to include representatives of both tolerance permissive and non-permissive outcomes. The general inability of modulated DC to achieve tolerance in such settings 1 indicates that non-permissive interactions currently dominate. Notwithstanding this, our findings have important implications for the development of DC for therapeutic application when tolerizing protocols will have to be sufficiently robust to mediate reprogramming in all patient scenarios.

Materials and methods

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

Mice

CBA/Ca (CBA), C57BL/6J (B6), CBA/Ca.RAG1−/− (CBA.RAG), C57BL/6J.RAG1−/− (B6.RAG) and TCR transgenic mice, A1(M).RAG1−/− (A1.RAG) 18 and Marilyn.RAG2−/− (Marilyn) 25 were bred and maintained in a specific pathogen-free facility at the Sir William Dunn School of Pathology, Oxford, UK. All procedures were conducted in accordance with the Home Office Animals (Scientific Procedures) Act of 1986 and received local ethical committee approval.

Preparation of BMDC

BM was cultured from 6- to 8–wk-old mice as described previously 33. Cells were fed on days 3 and 6, media removed on day 7 and lightly adherent DC harvested. Where indicated, 1 μg/mL of LPS (Escherichia coli, Sigma) was included for the final 18–20 h. For modulation, ∼20 ng/mL of murine IL-10 (transfected NS0 supernatant) or 10−7 M VD3 (Sigma) was added from day 3, or 2 ng/mL of human recombinant TGF-β1 (R&D Systems) from day 0. For recall assays, day 7 DC replated on day 6, were incubated at 37°C for 30 min with 10 μg/mL of mitomycin C (Sigma), washed and stored as frozen aliquots in 10% v/v DMSO.

Tolerance induction and skin grafting

Adoptive transfer of BMDC and skin grafting were performed as described previously 17. For Ab-induced tolerance, 1 mg of non-depleting anti-CD4 (YTS177.9.6) or IgG2a isotype control Ab was administered as a single dose on the day of grafting 26.

Flow cytometry

2G9 (anti-IAb/IEk), HL2 (anti-CD11c), 25–9–17 (anti-IAb), 14–4–4S (anti-IEk), 145-2C11 (anti-CD3), RM4-5 (anti-CD4), 7D4 (anti-CD25) and H1.2F3 (anti-CD69) (BD Bioscience) and FJK-16s (anti-Foxp3) (eBioscience). For BMDC and splenocytes, 10 μg/mL of 2.4G2 (anti-FcγIII/II) was included in the blocking buffer.

T-cell proliferation assays

Female responder splenic T cells purified by negative selection using the CD4+ T-cell isolation kit (Miltenyi) were plated at 2.5×104per well in 200 μL of RPMI containing 10% v/v FBS. Mitomycin C-treated male BMDC were used as stimulators, or alternatively, female BMDC were pulsed for 30 min at 37°C with 10–100 nM of appropriate HY-derived peptide: Dby-H2Ek, REEALHQFRSGRKPI (A1.RAG) 18, or Dby-H2Ab, NAGFNSNRANSSRSS (Marilyn) 25. Pulsed cells were washed three times before re-plating. At 48 h, co-cultures were pulsed with 0.5 μCi/well of tritiated thymidine (3H-TdR) (Amersham International) and assessed after a further 20–24 h. For recall responses, 5×104 nylon wool-purified splenic T cells from previously grafted mice were plated with female stimulator BMDC (1×104) in the presence of relevant Dby peptide. Prior to pulsing with 3H-TdR, 50 μL of supernatant was removed and IL-2 levels assessed by ELISA (Jes-1A12 and biotinylated Jes65H4, BD Pharmingen). To assess Ag proliferation in the absence of specific Ag, 96-well flat-bottomed plates were coated with anti-CD3 (145-2C11) and 2.5×104 AutoMacs-purified CD4+ T cells plated per well in 200 μL of BMDC medium without GM-CSF, but including 1 μg/mL of soluble anti-CD28 (37.51). Plates were pulsed after 48 h at 37°C using 0.5 μCi/well of 3H-TdR and assessed after 20–24 h.

SAGE and microarray pattern searching

SAGE libraries were generated as previously described or using Invitrogen's I–SAGE kits 33, 34. GEO accession numbers; GSM580, GSM767, GSM1681-2, GSM3677-79, GSM3681-6, GSM3824-29, GSM3832-7, GSM132450, GSM160043 and GSM269526, and http://www.sagenet.org/resources/3T3.htm. Mouse Known Gene SGC Oligo Set Arrays' (7524 oligonucleotide probes) (A-MEXP-54) were provided by The Human Genome Mapping Project Resource Centre, Cambridge, UK) and queried as E-MEXP-845 (EMBL-EBI ArrayExpress). Statistical assignment to idealized non-tolerant (BMDC+LPS) or tolerant (BMDC, BMDC-IL10±LPS, BMDC-TGF and BMDC-VD3) BMDC expression patterns was performed using SAGEClus software 35.

qPCR

Total RNA was reverse transcribed using random hexamer primers. Three biological replicates were performed as technical duplicates in 96-well format, using custom designed primers and probes (Eurogentech): Foxp3, CD3γ-chain and HPRT 17. Data were normalized to an endogenous control and expressed relative to a reference population using the comparative ΔΔCt method, essentially as described in ABI's User bulletin ♯2.

Acknowledgements

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

This work was supported by UK Medical Research Council grants G7904009 and G0300230l (S. P. C. and H. W.) and studentships (C. A. F. and S. F. Y.), the Rhodes (A. M. P.) and EP Abraham (S. P. C.) Trusts and the EU RISET consortium, project number: 512090 (H. W.).

Conflict of interest: The authors declare no financial or commercial conflict of interest.

References

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

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

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