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Keywords:

  • Costimulatory molecules;
  • DC;
  • Immunopathology;
  • Treg

Abstract

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

Classical DC (cDC) are required for efficient protective T-cell immunity. Moreover, recent data indicate that cDC also play a critical role in mediating homeostatic proliferation and maintenance of peripheral Treg. Here, we corroborate these findings by defining CD80/CD86 costimulation as an essential molecular component required for the cDC–Treg interactions. In contrast to earlier reports, the reduced Treg compartment of mice lacking cDC or selective CD80/86 expression on cDC, as such, did not render the respective animals prone to systemic lymphocyte hyperactivation or autoimmunity. Rather, we provide evidence that elevated immunoglobulin titers, as well as changes in T-cell subset prevalence and activation status are strictly associated with the nonmalignant myeloproliferative disorder triggered by the absence of cDC.


Introduction

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

Productive T-cell activation requires, in addition to the TCR stimulus, a second signal provided by costimulatory molecules, the best characterized of which are CD80 (B7-1) and CD86 (B7-2). CD80 and CD86, which are expressed mainly on B cells, DC and medullary thymic epithelial cells (mTEC) 1, are the only known ligands of CD28 and CTLA-4 receptors on T cells. Functions of CD28 and CTLA-4 are distinct with CD28 promoting T-cell activation and CTLA-4-negative regulating T-cell responses.

Peripheral self-tolerance and immune homeostasis are maintained, at least in part, by a delicate balance of T effector and Treg. CD25+CD4+ Treg, which arise spontaneously as the so-called natural Treg (nTreg) in the thymus, express the transcription factor forkhead box P3 (Foxp3) and can suppress the activation and proliferation of T lymphocytes in multiple ways. In addition, naïve T cells can also acquire Foxp3 expression in the periphery in the course of immune responses yielding inducible Treg with suppressive activity. Foxp3+ Treg, whether thymus derived or induced in the periphery, constitutively express both CTLA-4 and CD28 2. Moreover, CD80/86–CD28/CTLA4 interactions are required for the development, maintenance and function of Treg 3–6. Thus, the absence of CD80/86 results in a severe reduction of thymic Treg with no apparent changes in the percentages and distribution of conventional T-cell subsets 4. Furthermore, animals treated with B7-blocking antibodies and CD28-deficient mice display a markedly reduced Treg compartment 3–6. Available data suggest that both radio-resistant mTEC and BM-derived hematopoetic cells can deliver costimulatory signals that promote Treg generation in the thymus through CD80/86 interactions, with hematopoetic cells being more efficient 7.

In addition to their role in thymic Treg development, B7 interactions are also required to maintain the peripheral Treg compartment 3. Thus, administration of anti-CD80/86 antibodies reduces the percentage of peripheral Treg even in thymectomized mice lacking nTreg 4. Furthermore, adoptively transferred Treg show a reduced turnover in recipient mice subjected to B7-blockade 4, 8 and conversion of polyclonal naïve T cells into Foxp3+ Treg was found to be abrogated in B7-deficient recipient animals 9.

In vitro studies have revealed that BM culture-derived DC selected for high expression of CD86 are particularly effective in driving Treg proliferation 10 and that conversely DC isolated from CD80/86 double knockout mice poorly promote Treg division 4. Moreover, emerging evidence supports a direct correlation between DC numbers and the proliferation rate of peripheral Treg. Thus, Fms-like tyrosine kinase 3 ligand (Flt3L) treatment, which results in the in vivo expansion of classical DC (cDC) 11 leads to a concomitant increase in peripheral Treg 12, 13. Furthermore, it was recently demonstrated that the conditional ablation of cDC from otherwise intact animals results in reduced numbers and impaired homeostatic proliferation of peripheral Treg 13.

Here, we readdressed the role of cDC in the maintenance of peripheral Treg focusing on the role of CD80/86 costimulation. Using constitutive and conditional cDC ablation strategies, we established that peripheral Treg maintenance critically depends on the presence of cDC expressing CD80/86. Surprisingly however and defying earlier notions 13, 14, the reduction of Treg in animals lacking cDC as such was not inherently associated with lymphocyte activation. Rather than resulting from a tolerance failure, the autoinflammatory signatures reported for cDC-deficient mice are thus a consequence of the nonmalignant myeloproliferative disorder these animals develop.

Results

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

Reduced peripheral Treg numbers in the absence of DC

We and others recently reported that animals that constitutively lack cDC (CD11c-DTA mice) display normal percentages and numbers of thymic Foxp3+ Treg 14, 15, thereby establishing that DC are dispensable for the generation of nTreg. Moreover, CD11c-DTA mice retained functional peripheral Treg 15. However, closer examination of the blood circulation and LN of cDC-deficient animals and comparison to their littermate controls revealed a twofold reduction in the frequencies of Treg out of total CD4+ T cells, whose numbers are unaltered 15 (Fig. 1A). This reduction of peripheral Foxp3+ Treg was also observed upon conditional cDC ablation, as achieved through repetitive diphtheria toxin (DTx) treatment of [CD11c-DTR>WT] BM chimeras (Fig. 1B) 16, thereby confirming recent reports that established the critical role of cDC in promoting the homeostatic Treg proliferation 13, 17. Re-examination of Treg frequencies in cDC-deficient animals by staining for both Foxp3 and CD25 revealed a twofold reduction of Foxp3+CD25+ (double positive) Treg in all organs tested, including the spleen (Fig. 1C–E). Interestingly though, the decrease of splenic Foxp3+CD25+ Treg was uniquely associated with a concomitant elevation in the frequencies of Foxp3+CD25 (single positive) cells out of CD4+ T cells (Fig. 1E). This finding explains the reason why the splenic Foxp3+ T-cell compartment of cDC-deficient CD11c:DTA mice had, in the previous studies, appeared unaffected 14, 15. Collectively, these data establish that although cDC are not required for the generation of nTreg in the thymus, they are – in agreement with recent reports 13, 17 – critically involved in the maintenance of peripheral Foxp3+CD25+ Treg.

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Figure 1. Reduced peripheral Foxp3+CD25+ Treg in the absence of cDC (A) FACS-gating strategy used to define Foxp3+ Treg. Bars represent the percentages of Foxp3+ Treg out of blood CD4+ T cells of CD11c:DTA mice and littermate controls. n=5 for each group. p<0.001 using two-tail t-test. (B) Bars represent the percentages of Foxp3+ T cells out of blood CD4+ T cells of DTx/PBS-treated chimeras for 2 wk; (C–E) Bars represent the percentages of Foxp3+CD25+ and Foxp3+CD25 Treg out of CD4+ T cells in the mesenteric LN (C), blood (D) and spleen (E) of CD11c:DTA mice (black bars) and littermate control (white bars). Dot blots (E) represent CD25/Foxp3 staining gaiting of splenic CD4 T cells. Data are representative of three experiments.

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CD80/CD86 expression by cDC is crucial for peripheral Treg maintenance

To probe whether DC mediate peripheral Treg homeostasis in a B7-dependent mechanism and whether the reported peripheral Treg reduction in animals under CD28/B7 blockade 4 is hence specifically due to the lack of the B7 costimulation provided by cDC, we designed an experimental model in which the CD80/86 deficiency is restricted to CD11c-expressing cDC. The latter was achieved by generation of mixed BM chimeras through reconstitution of lethally irradiated WT recipient mice with an equal mixture of B7-deficient (CD80−/−CD86−/−) BM 18 and CD11c:DTA (CD45.1) BM 15. For controls, we included mice reconstituted with a mixture of B7 and WT (CD45.1) BM or CD11c:DTA, B7 and WT BM only (Fig. 2A). In the resulting mixed [B7/CD11c:DTA>WT ] BM chimeras, wt cDC are constantly ablated due to DTA expression. The cDC compartment of these animals thus consists exclusively of CD80−/−CD86−/− cDC. On the contrary, B cells and other hematopoetic cells in these animals are composed of both B7-proficient and -deficient cells, whereas nonhematopoetic cells, including the radio-resistant thymic epithelium, are exclusively of WT recipient genotype.

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Figure 2. DC-restricted B7 deficiency results in reduced peripheral Treg (A) Flow cytometric analysis of splenic cDC of the indicated chimeras. DC are gated as CD11chigh cells. Donor BM is indicated above the dot blots. Bar diagram summarizes the percentages of WT DC (CD45.1) and B7−/− DC (CD45.2) in the indicated chimeras. (B) Bars represent the percentages of Foxp3+ Treg out of single-positive CD4+ thymocytes in the indicated chimeras (cells were gated as shown in Fig. 1). (C) Bars represent the percentages of Foxp3+ Treg out of CD4+ T cells in the blood of indicated chimeras. (D) Bar diagrams represent the percentages of Foxp3+CD25+ Treg out of CD4+ T cells in the spleen of indicated chimeras. (E) [50% B7−/−/50% CD11c:DTR>WT] mix chimeras were treated with DTx/PBS for 10 days. Bar diagrams summarizing the percentages of Foxp3+ Treg out of CD4+ T cells in the blood (left) and spleen (right) of DTx/PBS-treated mix chimeras. Data show mean+SD, n=4 (B–E) and are representative of three (D) to four (A, B, C and E) independent experiments.

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Notably, the specific absence of CD80−/−CD86−/− from cDC in [B7/CD11c:DTA>wt] BM chimeras had no effect on the percentages of thymic Foxp3+ Treg out of single-positive CD4+ thymocytes (Fig. 2B). This corroborates earlier notions that mTEC and other, BM-derived APC can mediate the generation of nTreg in the thymus via B7 interactions 7, 19 and that thymic DC are dispensable for the generation of nTreg 14, 15. On the contrary, peripheral Foxp3+ Treg in [CD11c:DTA>WT] chimeras, constitutively lacking cDC, and [B7>WT] chimeras lacking CD80/CD86 expression on all BM-derived cells displayed markedly reduced Treg frequencies, when compared with [WT>WT] control chimeras (Fig. 2C and D). Moreover, importantly, the specific absence of CD80/CD86 on cDC, in the mixed [B7/CD11c:DTA>wt] BM chimeras, also resulted in more than twofold reduction of peripheral Foxp3+ Treg. In contrast, mixed [B7/WT>WT] BM chimeras retaining both B7-proficient and -deficient cDC displayed elevated percentages of Foxp3+ Treg, as compared with [B7/CD11c:DTA>wt] chimeras (Fig. 2C and D). It is worth noting that the only difference between these two groups of mixed BM chimeras is the absence of CD80/CD86-proficient cDC in [B7/CD11c:DTA>wt] chimeras. To substantiate our findings, we next generated mixed chimeras using BM of B7 mice (CD45.2) and CD11c-DTR mice (CD45.1) that allow for the conditional ablation of cDC 20. The resulting chimeras harbor a mixed DC-compartment consisting of DTx-sensitive WT DC and DTx-resistant B7 DC. DTx injection which leaves the chimeras only with CD80/CD86-deficient cDC resulted in a reduction of peripheral Treg (Fig. 2E). Collectively, these data establish that the peripheral Treg reduction observed in DC-ablated mouse models and B7 chimeras is due to the specific absence of the costimulatory molecules from cDC and that cDC mediate peripheral Foxp3+ Treg maintenance via CD80 and CD86 (B7) interactions.

The reduced frequencies of peripheral Treg caused by DC deficiency do not result in autoimmunity

Foxp3+ Treg are functionally defined by their suppressive activity on effector T cells directed against foreign and self-antigens 21. The observed reduced Treg compartment of mice lacking cDC or selected CD80/86 expression on cDC could hence render these animals prone to develop autoimmunity. Indeed, CD11c-DTA mice, which as shown above have a Treg deficiency, display the features of systemic lymphocyte activation, such as the accumulation of cells with memory T-cell phenotype (CD62LloCD44hi) (Fig. 3A), prevalence of Th17 and Th1 cells (Fig. 3B) and elevated IgG1, but not IgM serum titers (Fig. 3C). Notably, Ohnmacht et al. interpreted these findings as an indication of a general tolerance failure in cDC-less mice resulting in fatal autoimmunity 14. Furthermore, animals transiently depleted of cDC have also been reported to display elevated Th1 and Th17 cells, supporting the notion of impaired peripheral tolerance 13. In the latter study, the authors specifically suggested that these features result from the impaired Treg compartment of cDC-depleted animals 13. However, as we recently reported 15, CD11c:DTA mice that constitutively lack cDC also develop a progressive nonmalignant myeloproliferative disorder, driven by elevated systemic Flt3L levels. In the absence of measurable T-cell autoreactivity in DC-depleted mice 15, we hence had interpreted their above-mentioned features of lymphocyte activation, as consequences of the pathological systemic accumulation of myeloid cells, rather than as a result of a breakage of adaptive immune tolerance. Given our present finding that CD11c:DTA mice harbor an impaired Treg compartment (Fig. 1), we decided to revisit this issue and investigate whether the Treg deficiency resulting from cDC ablation causes lymphocyte hyperactivation or autoimmunity. Specifically, we took advantage of the fact that the above-mentioned [B7/CD11c:DTA>wt] BM chimeras display a similar reduction of their Treg compartment, as DC- or B7-deficient animals, but due to the presence of CD80−/−CD86−/− cDC do not develop a myeloproliferative disorder (Fig. 4A). Importantly, [B7/CD11c:DTA>wt] chimeras lacked all “autoimmune signatures” previously reported for CD11c:DTA and DTx-treated CD11c-DTR mice 13–15. This included the elevated frequencies of CD4+CD62LloCD44hi “memory” T cells (Fig. 4B), the increased prevalence of IFN-γ- and IL-17-producing cells (Fig. 4C) and the elevated IgG1 titers (Fig. 4D). These data thus establish that the “autoimmune signatures” of cDC-deficient mice are strictly associated with the development of the Flt3L-driven myeloproliferation and hence likely a consequence thereof. In support of this notion, we observed that a myeloid expansion induced by inoculation of WT mice with Flt3L-secreting tumor cells 22 also resulted in the accumulation of CD62LloCD44hi T cells (Fig. 4E). Collectively, we conclude that the reduction of peripheral Treg resulting from their lack of B7-expressing cDC plays no causative role in the lymphocyte hyperactivation observed in these animals. Rather, the reported autoimmune deviations of cDC-less animals 13, 14 are related to their development of a chronic myeloproliferative disorder.

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Figure 3. Phenotype of lymphocyte activation in CD11c:DTA mice. (A) Flow cytometry analysis of CD4+ T cells isolated from the spleen of 5-month-old CD11c:DTA mice or littermate controls. Bars represent the percentages of activated CD4+ T cells (CD62LlowCD44high) out of CD4+ T cells. n=3 for each group. Data show mean+SD and are representative of four independent experiments. (B) Flow cytometry analysis of mesenteric LN CD4+ T cells (gated as in Fig. 3A). Bars represent the frequencies of IFN-γ- or IL-17-expressing cells out of CD4+ T cells in the mesenteric LN of 5-month-old CD11c:DTA mice and littermate controls. Data show mean+SD and are representative of three independent experiments. (C) Serum IgM (left) and IgG1 (right) titers of CD11c:DTA mice and age-matched littermate controls as determined by ELISA. Data show mean+SD and are representative of four independent experiments.

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Figure 4. The reduced frequencies of peripheral Treg caused by DC deficiency do not result in autoimmunity. (A) Bars represent the percentages of CD11b+Gr-1+ myeloid cells out of total splenocytes in the indicated BM chimeras. Donor BM is indicated below the bars. Data show mean+SD, n=4, and are representative of four independent experiments. (B) Bar diagrams represent the frequencies of activated CD4+ T cells (CD62Llow CD44high) out of CD4+ T cell in the spleen of the indicated BM chimeras. Donor BM is indicated below the bars. Gating as indicated in Fig. 3A. Data show mean+SD, n=4, and are representative of three independent experiments. (C) IFN-γ-expressing cells (left) and IL-17-expressing cells (right) out of total CD4+ T cells in the mesenteric LN of the indicated BM chimeras. Donor BM is indicated below the bars. Gating as indicated in Fig. 3B. Data show mean+SD, n=4, and are representative of two independent experiments. (D) Serum IgG1 titers of indicated chimeras as determined by ELISA. Data show mean+SD, n=4, and are representative of three independent experiments. (E) Flow cytometry analysis of splenic CD4+ T cells from C57BL/6 mice bearing a tumor-secreting Flt3L. Numbers indicate the mean frequencies of CD62Llow CD44high activated cells out of CD4+ T cells. Bars represent the percentages of neutrophils (CD11b+Gr-1high) and monocytes (CD11b+Gr-1low/−) out of total splenic cells. Data show mean+SD, n=3, and are representative of three independent experiments.

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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

Here, we established that expression of the costimulatory molecules CD80 and CD86 by cDC is required for peripheral Treg maintenance. As such, our studies complement a recent study demonstrating that cDC control Treg homeostasis in dependence of MHC II expression 13. Using CD80/CD86 mutant animals and a strategy that restricts the B7 deficiency to cDC, we show here that cDC also have to provide a critical costimulatory signal to the Treg.

Animals that constitutively lack cDC display features of systemic lymphocyte activation including hypergammaglobulinemia, the accumulation of CD62LloCD44hi T cells and an increased prevalence of Th17 and Th1 cells 14, 15. Ohnmacht et al. interpreted these findings as an indication of a general tolerance failure in these animals resulting in fatal autoimmunity 14. Furthermore, after establishing that cDC are required for Treg homeostasis, Darrasse-Jeze et al. suggested that the elevation in Th1 and Th17 in cDC-depleted animals is a result of their impaired Treg compartment 13. However, as we recently reported 15, constitutive and conditional ablation of cDC triggers a systemic elevation of the growth factor Flt3L causing a progressive nonmalignant myeloproliferative disorder. Here, we show that the feedback loop that links the peripheral cDC compartment to myelogenesis is not mediated through CD80/86 interaction since animals that exclusively harbored B7-deficient cDC did not develop the myeloproliferation. We had previously interpreted the lymphocyte activation in cDC-depleted mice as a consequence of the systemic pathological accumulation of myeloid cells, rather than as a result of a breakage of adaptive immune tolerance. In support of this notion, we had despite major efforts failed to detect T-cell autoreactivity in these animals 15. Taking advantage of mice that harbor the cDC-restricted B7 deficiency and display a reduction of Treg without associated myeloproliferation, we show in thid study that the Treg reduction resulting from impaired cDC/T-cell crosstalk does as such not result in lymphocyte hyperactivation. Rather than reflecting a tolerance failure or autoimmunity, our results suggest that the latter is a secondary consequence of the Flt3L-driven myeloproliferative disorder observed in cDC-deficient animals. This notion is supported by the fact that other animals displaying myeloproliferative disorders, such as IRF8-deficient mice, have also been reported to suffer from hypergammaglobulemias 23. Moreover, Flt3L-treated mice which were reported to develop an MPD similar to the one observed in cDC-deficient animals also display an elevation in the frequencies of CD62LloCD44hi memory phenoptype CD4+ T cells although they retain elevated DC number and normal Treg numbers.

It could be argued that T-lymphocyte activation and hence the priming of potentially autoreactive CD4+ T cells could be impaired in the mixed [B7/CD11c:DTA>WT ] BM chimeras due to the absence of cDC-derived costimulation. However, as shown in this study and reported by Ohnmacht et al. 14, activation of T cells can occur in the complete absence of cDC. Thus cells other than cDC, i.e. MHC class II+ hematopoietic APC, including plasmacytoid DC 15, B cells and macrophages, as well as nonhematopoietic MHC class II+ enterocytes seem sufficient to activate T lymphocytes in particular under pathological conditions.

Notably, our data do not dispute the role of Treg in the control of autoreactive T-cell immunity, as for instance established by direct Treg ablation strategies 24–26. Rather, they discriminate these systems from the partial Treg impairment induced by cDC deficiencies, which seems to be well buffered and tolerated by the organism. We believe our finding should spur a general re-evaluation of current classifications of the spontaneous immune disorders observed in mouse models. In the clinic, many diseases, previously labeled “autoimmune” are gradually redefined due to the lack of MHC and autoantibody associations. According to a suggested refined nomenclature 27, autoimmunity should be seen as a result of aberrant B- and T-cell responses in primary and secondary lymphoid organs breaking tolerance, with the development of immune reactivity toward native self-antigens. Adaptive immune responses play a predominant role in these diseases. In contrast, self-directed inflammation, in which local factors at predisposed sites lead to activation of innate immune cells, such as macrophages and neutrophils, resulting in target tissue damage, should be considered autoinflammation. Examples of the latter are the disturbed homeostasis of canonical cytokine cascades (as in periodic fevers 28 and aberrant bacterial sensing or barrier functions (as in Crohn's disease)). Drastic systemic aberrations, such as the progressive Flt3L-driven myeloid proliferative disorder observed in cDC-less mice 15, likely predispose to site-specific inflammation, which is initially independent of adaptive immune responses. Along these lines, it is noteworthy that neutrophils have been reported to express B-cell activating factor (BAFF) 29 and that mere BAFF overexpression in mice results in a SLE-like syndrome 30. Interestingly and in accordance with the notion that their disorder could have an innate origin, the spontaneous disease manifestations reported for cDC-deficient animals 13, 14 are restricted to the intestine, suggesting the microflora-driven processes that might be amenable to antibiotic treatment. While in the clinic, the discrimination of autoinflammation and autoimmunity has important implications in the choice of the appropriate therapeutic strategies, such as anti-lymphocyte or anti-cytokine treatment, we believe it could also advance our understanding of animal disease models and hence benefit their use in respective preclinical studies.

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

The following mice were used in this study: C57BL/6 mice, CD80/86−/− 18 CD11c-DTR transgenic (B6.FVB-Tg Itgax-DTR/GFP 57Lan/J) mice carrying a transgene encoding a human DTR-GFP fusion protein under the control of the murine CD11c promoter 15; CD11c-Cre mice 31, R26-DTA mice 32 and R26-DTA mice were crossed with CD11c-Cre transgenic mice to generate CD11c-Cre:DTA mice 15. For conditional DC ablation [CD11c-DTR>wt], BM chimeras were inoculated intraperitoneally every second day for 2 wk with 16 ng DTx/g body weight. For BM chimera generation, recipient mice were lethally irradiated with a 950 rad dose and a day later i.v. injected with 5×106 BM cells isolated from donors femora and tibiae. BM recipients were then allowed to rest for 8 wk before use. All mice were maintained under specific pathogen-free conditions and handled under protocols approved by the Weizmann Institute Animal Care Committee according to international guidelines.

Flow cytometry analysis

Staining reagents used in this study included the PE-coupled antibodies anti-MHC II, CD25, CD62L, CD8, CD11b, CD115, CD80, IL-17; the biotinylated antibodies: anti CD45.1, CD4, CD3; the APC-coupled antibodies: anti CD11c, CD4, CD44, IFN-γ, CD19 and Gr-1 (Ly6C/G); and PerCP-coupled streptavidin. Foxp3 intracellular staining was performed according to the manufacturer's protocol (eBioscience 77-5775-40). Unless indicated otherwise, the reagents were obtained from eBioscience or Biolegend. The cells were analyzed on a FACS Calibur cytometer (Becton-Dickinson) using CellQuest software (Becton-Dickinson).

Intracellular cytokine staining

Cells obtained from mesenteric LN were incubated at 37C for 4 h in 10% FBS DMEM medium with 50 ng/mL PMA (Sigma-Aldrich) and 1 μg/mL ionomyicin (Sigma-Aldrich). Brefeldin A (5 μg/mL, Sigma-Aldrich) was added after 2 h. Cells were resuspended in fixation/permeabilization solution (Cytofix/Cytoperm kit, BD). Intracellular cytokine staining using anti-IL-17 and anti-IFN-γ was performed according to the manufacturer's protocol.

ELISA

Serum immunoglobulin isotypes were determined using commercial ELISA antibodies (SouthernBiotech).

In vivo exposure to Flt3L

C57BL/6 mice were inoculated with B16 tumor cells (3×106) that had been manipulated to overexpress Flt3L 22.

Statistical analysis

All statistics were generated using a Student's t-test. All error bars in diagrams, and numbers following a ± sign, are standard deviations.

Acknowledgements

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

The authors thank all lab members of the Jung laboratory for helpful discussions. This work was supported by the Israel Science Foundation (ISF) and the Yeda-Sela Center for Basic Research.

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
  • 1
    Sharpe, A. H. and Freeman, G. J., The B7-CD28 superfamily. Nat. Rev. Immunol. 2002. 2: 116126.
  • 2
    Wing, K., Onishi, Y., Prieto-Martin, P., Yamaguchi, T., Miyara, M., Fehervari, Z., Nomura, T. and Sakaguchi, S., CTLA-4 control over Foxp3+regulatory T cell function. Science 2008. 322: 271275.
  • 3
    Salomon, B., Lenschow, D. J., Rhee, L., Ashourian, N., Singh, B., Sharpe, A. and Bluestone, J. A., B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+immunoregulatory T cells that control autoimmune diabetes. Immunity 2000. 12: 431440.
  • 4
    Tang, Q., Henriksen, K. J., Boden, E. K., Tooley, A. J., Ye, J., Subudhi, S. K., Zheng, X. X. et al., Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+regulatory T cells. J. Immunol. 2003. 171: 33483352.
  • 5
    Lohr, J., Knoechel, B., Kahn, E. C. and Abbas, A. K., Role of B7 in T cell tolerance. J. Immunol. 2004. 173: 50285035.
  • 6
    Tai, X., Cowan, M., Feigenbaum, L. and Singer, A., CD28 costimulation of developing thymocytes induces Foxp3 expression and regulatory T cell differentiation independently of interleukin 2. Nat. Immunol. 2005. 6: 152162.
  • 7
    Proietto, A. I., van Dommelen, S., Zhou, P., Rizzitelli, A., D'Amico, A., Steptoe, R. J., Naik, S. H. et al., Dendritic cells in the thymus contribute to T-regulatory cell induction. Proc. Natl. Acad. Sci. USA 2008. 105: 1986919874.
  • 8
    Golovina, T. N., Mikheeva, T., Suhoski, M. M., Aqui, N. A., Tai, V. C., Shan, X., Liu, R. et al., CD28 costimulation is essential for human T regulatory expansion and function. J. Immunol. 2008. 181: 28552868.
  • 9
    Liang, S., Alard, P., Zhao, Y., Parnell, S., Clark, S. L. and Kosiewicz, M. M., Conversion of CD4+CD25− cells into CD4+CD25+regulatory T cells in vivo requires B7 costimulation, but not the thymus. J. Exp. Med. 2005. 201: 127137.
  • 10
    Yamazaki, S., Iyoda, T., Tarbell, K., Olson, K., Velinzon, K., Inaba, K. and Steinman, R. M., Direct expansion of functional CD25+CD4+ regulatory T cells by antigen-processing dendritic cells. J. Exp. Med. 2003. 198: 235247.
  • 11
    Maraskovsky, E., Daro, E., Roux, E., Teepe, M., Maliszewski, C. R., Hoek, J., Caron, D. et al., In vivo generation of human dendritic cell subsets by Flt3 ligand. Blood 2000. 96: 878884.
  • 12
    Swee, L. K., Bosco, N., Malissen, B., Ceredig, R. and Rolink, A., Expansion of peripheral naturally occurring T regulatory cells by Fms-like tyrosine kinase 3 ligand treatment. Blood 2009. 113: 62776287.
  • 13
    Darrasse-Jeze, G., Deroubaix, S., Mouquet, H., Victora, G. D., Eisenreich, T., Yao, K. H., Masilamani, R. F. et al., Feedback control of regulatory T cell homeostasis by dendritic cells in vivo. J. Exp. Med. 2009. 206: 18531862.
  • 14
    Ohnmacht, C., Pullner, A., King, S. B., Drexler, I., Meier, S., Brocker, T. and Voehringer, D., Constitutive ablation of dendritic cells breaks self-tolerance of CD4 T cells and results in spontaneous fatal autoimmunity. J. Exp. Med. 2009. 206: 549559.
  • 15
    Birnberg, T., Bar-On, L., Sapoznikov, A., Caton, M. L., Cervantes-Barragan, L., Makia, D., Krauthgamer, R. et al., Lack of conventional dendritic cells is compatible with normal development and T cell homeostasis, but causes myeloid proliferative syndrome. Immunity 2008. 29: 986997.
  • 16
    Zaft, T., Sapoznikov, A., Krauthgamer, R., Littman, D. R. and Jung, S., CD11chigh dendritic cell ablation impairs lymphopenia-driven proliferation of naive and memory CD8+T cells. J. Immunol. 2005. 175: 64286435.
  • 17
    Suffner, J., Hochweller, K., Kuhnle, M. C., Li, X., Kroczek, R. A., Garbi, N. and Hammerling, G. J., Dendritic cells support homeostatic expansion of Foxp3+regulatory T cells in Foxp3. LuciDTR mice. J. Immunol. 2010. 184: 18101820.
  • 18
    Borriello, F., Sethna, M. P., Boyd, S. D., Schweitzer, A. N., Tivol, E. A., Jacoby, D., Strom, T. B. et al., B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation. Immunity 1997. 6: 303313.
  • 19
    Hinterberger, M., Aichinger, M., da Costa, O. P., Voehringer, D., Hoffmann, R. and Klein, L., Autonomous role of medullary thymic epithelial cells in central CD4(+) T cell tolerance. Nat. Immunol. 2010. 11: 512519.
  • 20
    Jung, S., Unutmaz, D., Wong, P., Sano, G., De los Santos, K., Sparwasser, T., Wu, S. et al., In vivo depletion of CD11c(+) dendritic cells abrogates priming of CD8(+) T cells by exogenous cell-associated antigens. Immunity 2002. 17: 211220.
  • 21
    Littman, D. R. and Rudensky, A. Y., Th17 and regulatory T cells in mediating and restraining inflammation. Cell 2010. 140: 845858.
  • 22
    Mach, N., Gillessen, S., Wilson, S. B., Sheehan, C., Mihm, M. and Dranoff, G., Differences in dendritic cells stimulated in vivo by tumors engineered to secrete granulocyte-macrophage colony-stimulating factor or Flt3-ligand. Cancer Res. 2000. 60: 32393246.
  • 23
    Holtschke, T., Lohler, J., Kanno, Y., Fehr, T., Giese, N., Rosenbauer, F., Lou, J. et al., Immunodeficiency and chronic myelogenous leukemia-like syndrome in mice with a targeted mutation of the ICSBP gene. Cell 1996. 87: 307317.
  • 24
    Kim, J. M., Rasmussen, J. P. and Rudensky, A. Y., Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat. Immunol. 2007. 8: 191197.
  • 25
    Lahl, K., Loddenkemper, C., Drouin, C., Freyer, J., Arnason, J., Eberl, G., Hamann, A. et al., Selective depletion of Foxp3+regulatory T cells induces a scurfy-like disease. J. Exp. Med. 2007. 204: 5763.
  • 26
    Feuerer, M., Shen, Y., Littman, D. R., Benoist, C. and Mathis, D., How punctual ablation of regulatory T cells unleashes an autoimmune lesion within the pancreatic islets. Immunity 2009. 31: 654664.
  • 27
    McGonagle, D., Aziz, A., Dickie, L. J. and McDermott, M. F., An integrated classification of pediatric inflammatory diseases, based on the concepts of autoinflammation and the immunological disease continuum. Pediatr Res. 2009. 65: 38R45R.
  • 28
    McDermott, M. F., Aksentijevich, I., Galon, J., McDermott, E. M., Ogunkolade, B. W., Centola, M., Mansfield, E. et al., Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes. Cell 1999. 97: 133144.
  • 29
    Scapini, P., Bazzoni, F. and Cassatella, M. A., Regulation of B-cell-activating factor (BAFF)/B lymphocyte stimulator (BLyS) expression in human neutrophils. Immunol. Lett. 2008. 116: 16.
  • 30
    Khare, S. D., Sarosi, I., Xia, X. Z., McCabe, S., Miner, K., Solovyev, I., Hawkins, N. et al., Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc. Natl. Acad. Sci. USA 2000. 97: 33703375.
  • 31
    Caton, M. L., Smith-Raska, M. R. and Reizis, B., Notch-RBP-J signaling controls the homeostasis of CD8− dendritic cells in the spleen. J. Exp. Med. 2007. 204: 16531664.
  • 32
    Brockschnieder, D., Pechmann, Y., Sonnenberg-Riethmacher, E. and Riethmacher, D., An improved mouse line for Cre-induced cell ablation due to diphtheria toxin A, expressed from the Rosa26 locus. Genesis 2006. 44: 322327.

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

See accompanying commentary:http://dx.doi.org/10.1002/eji.201041335

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