The role of suppressor of cytokine signaling 1 as a negative regulator for aberrant expansion of CD8
+ dendritic cell subset
Jun Tsukada1,2,
Akemi Ozaki1,
Toshikatsu Hanada3,
Takatoshi Chinen3,
Ryo Abe2,
Akihiko Yoshimura3 and
Masato Kubo1
1 Laboratory for Signal Network, Research Center for Allergy and Immunology (RCAI), RIKEN Yokohama Institute, Suehiro-cho 1-7-22, Tsurumi, Yokohama, Kanagawa 230-0045, Japan
2 Division of Immunobiology, Research Institute for Biological Sciences, Tokyo University of Science, 2669 Yamazaki, Noda City, Chiba 278-0022, Japan
3 Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Umaide, Higashi-hu Fukuoka 812-8582, Japan
Correspondence to: M. Kubo; E-mail: raysolfc{at}rcai.riken.jp
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Abstract
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The suppressor of cytokine signaling (SOCS) 1 is a negative regulator in multiple cytokine-related aspects to maintain immunological homeostasis. Here, we studied a role of SOCS1 on dendritic cell (DC) maturation in the mice lacking either TCR
chain or CD28 in SOCS1-deficient background, and found that the SOCS1 could restore acute phase of inflammatory response in SOCS1-deficient mice. The CD11c+ CD8 DC population in freshly isolated splenic DCs from normal mice highly expressed SOCS1. However, in SOCS1-deficient environment, the proportion of CD8
+ DCs (CD8 DCs) noticeably increased without affecting the cell number of conventional and plasmacytoid DC populations. This population revealed the CD11cdull CD8
+ CD11b CD45RA B220 phenotype, which is a minor population in normal mice. Localization of the abnormal CD8 DCs in splenic microenvironments was mainly restricted to deep within red pulp. The CD8 DCs secrete a large amount of IFN-
, IL-12 and B lymphocyte stimulator/B cell activation factor of the tumor necrosis factor family in response to LPS and CpG stimulation. This is responsible for the development of DC-mediated systemic autoimmunity in the old age of SOCS1-deficient mice. Moreover, the CD8 DC subsets expressed more indoleamine 2,3-dioxygenase and IL-10, and hence inhibit the allogeneic proliferative T cell response and antigen-induced Th1 responses. Therefore, SOCS1 expression during DC maturation plays a role in surveillance in controlling the aberrant expansion of abnormal DC subset to maintain homeostasis of immune system.
Keywords: antigen presentation, CD8, dendritic cell, SOCS1
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Introductions
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Dendritic cells (DCs) are very effective antigen-presenting cells (APCs), and play a crucial role in triggering the immune system against infection-causing agents and cancer. It has been commonly accepted that the DCs are produced in the bone marrow and migrate into almost all tissues via blood (1). The DC changes its function from capturing antigens to presenting them during differentiation from immature cells to mature DCs through various stages. The DC maturation can be triggered by pathogens through direct interaction with receptors, such as Toll-like receptors (TLRs) (2). Mature DCs have potent ability to prime antigen-specific naive helpers and CTLs in secondary lymphoid organs (35).
Characterizations of DC development, maturation and subsets are based on phenotypes, functional potential and microenvironmental localization, which are capable of inducing distinct types of responses (6). Mature CD11c+ DCs in mouse spleen can be classified into two DC subsets on the basis of CD8
expression. The functional difference between CD8
and CD8
+ sub-populations has been extensively studied recently. Previous reports have demonstrated their distinct biological functions, such as the ability to induce Th1 and Th2 responses (7, 8). CD8
+ DC subset has originally been reported as tolerogenic DC, and acts as a regulatory or stimulatory DC, depending on conditions and their states of activation and maturation (9, 10). Recent reports have accumulated the evidences that torelogenic DCs have a significant role in the induction of peripheral tolerance through IL-10 production, co-stimulation molecule deficiency or expression of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) (9, 11, 12).
DCs were originally thought to be derived from myeloid precursors in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF). Evidences from in vitro differentiation assays indicate that the DC maturation is accomplished in bone marrow cultures in the presence of GM-CSF with tumor necrosis factor-
or IL-4 (1315). This system leads to the differentiation of DCs with phenotypic and functional characteristics that are similar to those described for human monocyte-derived DCs. These previous observations have shown the indispensable role of cytokine signaling in the development of DCs.
Suppressor of cytokine signaling (SOCS) molecules belong to intracellular proteins that inhibit Janus kinase (JAK), signal transduction and activators of transcription (STAT) pathway (1622). The SOCS1 is an immediately induced gene upon cytokine stimulations, such as IFN-
(23, 24), and binds to all family members of JAKs in SH2-phosphotyrosine-based interaction. A disruption of SOCS1 gene results in stunted growth, and all mice born with severe lymphopenia, activation of peripheral T cells, fatty degeneration and liver necrosis will die within 3 weeks (25, 26). This lethality in early age can be rescued in double-deficient mice with IFN-
, STAT1 or recombination activating gene 2 (RAG2) (27, 28). These results suggest that the SOCS1 deficiency causes abnormal expansion of IFN-
-producing T and NKT cells, and subsequently results in acute liver injuries. Our recent study demonstrated that the lymphocyte-specific SOCS1-transgenic mice on a SOCS1 KO background (SOCS1 KO-Tg) could survive the neonatal death. SOCS1 KO-Tg develops systemic lupus erythematosus-like autoimmune diseases, including dermatitis, renal accumulation of antigenantibody complex, high levels of Ig and anti-DNA antibodies in sera in their later months (29). We observed that the abnormal DC population in the absence of SOCS1 was highly activated, and it highly expressed B cell growth factor, B lymphocyte stimulator/B cell activation factor of the tumor necrosis factor family (BLyS/BAFF), and a proliferation-inducing ligand (APRIL), which led to expansion of the B cells secreting autoreactive antibody.
These observations raise the question of whether or not the SOCS1 expression has intrinsic role in the development of DC subsets. SOCS1/CD28 doubly deficient (SOCS1/CD28 DKO) mice could rescue the lethality with acute inflammatory reaction in the younger SOCS1 KO mice. This is attributed to a lack of primary T cell activations in the CD28-deficient mice. The SOCS1/CD28 DKO mice exhibited no pathological symptom for inflammatory reaction until they were older than 3 months. Therefore, these mice allow us to study an intrinsic role of SOCS1 on DC maturation in the absence of inflammatory symptom. In the present study, we analyzed the freshly isolated splenic DC sub-populations because SOCS1 KO-derived DCs have high sensitivity against any cytokine stimulation. The splenic DC population in the absence of SOCS1 expression exhibited the expansion of abnormal CD8
DC population that secreted IFN-
and IL-12 in response to LPS and CpG, resulting in enhancement of Th1-dominant immune responses. This DC population inhibited class II-mediated immune response. We also discussed the physiological significance of SOCS1 expression profile in DC populations in order to maintain immune surveillance.
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Methods
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Mice
SOCS1 KO and CD28 KO mice have been previously established (28, 30) and back-crossed onto a C57BL/6 (B6) background for over 10 generations. SOCS1/CD28 DKO mice were generated by cross-breeding between SOCS1 KO mice and CD28 KO mice. SOCS1/TCR DKO mice were described previously (29). OT-II Tg mice on C57BL/6 background were kindly provided by K. Yui (Nagasaki University). Female mice younger than 10 weeks were used for all experiments in the present study.
Purification and characterization of DCs
DCs were isolated from spleen or thymus with anti-CD11c-Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), and CD11c+ cells were positively enriched by a MACS separation column. Sub-population of DCs was analyzed by the combination of the following antibodies: anti-CD4FITC, anti-CD8
FITC or PE, anti-CD11bFITC, anti-CD40FITC, anti-CD45RAFITC, anti-CD80FITC, anti-CD86FITC, anti-I-AbFITC and anti-CD11cbiotin (BD Biosciences PharMingen, San Diego, CA, USA). Biotinylated mAbs were detected with streptavidinPE or Cy-Chrome, and subsequently stained cells were analyzed.
Purification of T cell and antigen-induced Th differentiation
The CD4+ T cells were enriched from spleen of either BALB/c or OT-II Tg mice. Splenocytes were incubated with anti-CD8 mAb (3155) and were placed on the anti-mouse Ig-coated plate (Cappel, Aurora, OH, USA) to eliminate the CD8+ T and B cells. Bone marrow-derived DCs (BM-DCs) were prepared from bone marrow suspension from normal C57BL/6 mice and cultured in the presence of GM-CSF for 7 days. CD4+ T cells (1 x 106 ml1) from OT-II Tg mice were stimulated with ovalbumin peptide [OVA323339, Loh15 (6 µM)] in the context of syngeneic BM-DCs (1 x 105 ml1). After 5 days, cells were harvested and re-stimulated with anti-TCR mAb for 6 h in the presence of 4 µM monensin (SigmaAldrich, St Louis, MO, USA). Intracellular cytokine staining was carried out with FITC-labeled anti-IFN-
mAb (XNG1.2) and PE-labeled anti-IL-4 mAb (11B11) (31).
Immunohistochemical analysis
Tissue sections were prepared following Kawamoto's method, a method for preparing 2- to 50-µm-thick fresh-frozen sections of large samples and undecalcified hard tissues. Briefly, spleens were immediately frozen in 3% carboxymethyl cellulose gel. Sections of 4-µm thickness were cut and fixed in ice-cold methanol, and then washed with PBS. Slides were blocked with 1% normal mouse serum, 1% FCS and 1% BSA in PBS, and they were washed and then further blocked with anti-FcR antibody (2.4G2). After washing, slides were exposed to PEanti-mouse CD11c mAb and FITCanti-CD8
mAb, and incubated at 4°C overnight. Confocal imaging was performed using spectral confocal scanning system, Leica TCS SP2 (Leica Microsystems, Wetzlar, Germany).
Quantitative reverse transcriptionPCR analysis
DCs were isolated from spleen with anti-CD11c-Microbeads and stimulated with CpG (1 µM). Total cellular RNA was isolated from the DC preparations by Trysol (SigmaAldrich). Real-time quantitative reverse transcriptionPCR analysis was performed using ABI PRISM 7700 sequence detection systems (Applied Biosystems, University Park, IL, USA). The following primers were used for PCR of ß-actin, SOCS1, SOCS3, BAFF, TLR4, TLR9, CCR7 and IDO: ß-actin, sense 5'-ACTATTGGCAACGAGCGGTTC-3' and anti-sense 5'-GGATGCCACAGGATTCCATACC-3'; SOCS1, sense 5'-CACCTTCTTGGTGCGCG-3' and anti-sense 5'-AAGCCATCTTCACGCTGAGC-3'; SOCS3, sense 5'-CTTCAGCATCTCTGTCGGAAGA-3' and anti-sense 5'-ATCGTACTGGTCCAGGAACTCC-3'; BAFF, sense TGCTATGGGTCATGTCATCCA-3' and anti-sense 5'-GGCAGTGTTTTGGGCATATTC-3'; TLR4, sense 5'-ACTGGGTGAGAAATGAGCTGGT-3' and anti-sense 5'-GAATAAAGTCTCTGTAGTGAAGGCAGA-3'; TLR9, sense 5'-ACTTGATGTGGGTGGGAATTG-3' and anti-sense 5'-GCCACATTCTATACAGGGATTGG-3'; CCR7, sense 5'-TGCTGCGTCAACCCTTTCT-3' and anti-sense 5'-AGTCCTTGAAGAGCTTGAAGAGGT-3'; IDO, sense 5'-GAGAAAGCCAAGGAAATTTTTAAGAG-3' and anti-sense 5'-GATATATGCGGAGAACGTGGAAA-3'. FAM (6-carboxyfluorescein)-labeled probes were used as target hybridization probes for ß-actin, SOCS1, SOCS3, BAFF, TLR4, TLR9, CCR7 and IDO: ß-actin, 5'-CCTGAGGCTCTTTTCCAGCCTTCCTTCT-3'; SOCS1, 5'-TCGCCAACGGAACTGCTTCTTCG-3'; SOCS3, 5'-AACGGCCACCTGGACTCCTATGAGAAAG-3'; BAFF, 5'-AGCCTGGTGACCCTGTTCCGATGTATTC-3'; TLR4, 5'-CTTACCGGGCAGAAGGAAGTAGCACT-3'; TLR9, 5'-CGTCGCTGCGACCATGCC-3'; CCR7, 5'-TGCATGATGGGCGCAGACTGG-3', and IDO, 5'-TGCGTGACTTTGTGGACCCAGACAC-3'. Each mRNA quantity was normalized by the level of ß-actin.
Measurement of cytokine production by ELISA assays
Spleen-derived CD11c+ DCs were cultured in RPMI1640 supplemented with 10% fetal bovine serum in the presence of GM-CSF (20 ng ml1) and stimulated with either LPS (100 ng ml1) or CpG (1 µM), and the culture supernatants were harvested after 24 h. The concentration of IL-12, IFN-
and IL-10 was measured by ELISA. Briefly, the supernatants were applied on the plastic plate coated with captured specific antibodies (BD Biosciences PharMingen). After washing, the plate was probed with biotin-conjugated detection antibodies and HRP-conjugated streptavidin (Zymed, San Francisco, CA, USA) and developed with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Kirkegard and Perry Laboratories, Gaithersburg, MD, USA). The 405-nm absorbance was measured by spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA).
For cytokine array of inflammatory cytokines and chemokines production in splenic DCs, CD11c+ population was stimulated with CpG (1 µM) in the presence of GM-CSF (20 ng ml1). The culture supernatants were harvested after 24 h, and cytokine array were carried out with Mouse Cytokine Antibody Array (RayBio, Norcross, GA, USA).
Proliferation assays
The CD4+ T cells were enriched from spleen of BALB/c mice and were placed on the anti-mouse Ig-coated plate after incubation with anti-CD8 mAb (3155). BALB/c CD4+ T cells were cultured with irradiated splenic DCs of B6 mice as allogeneic stimulator for 48 h. Cells were pulsed with 1 mCi of [3H]thymidine ([3H]TdR) (Amersham Biosciences, Piscataway, NJ, USA) for last 8 h, and proliferative responses were assessed by incorporation of [3H]TdR.
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Result
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Aberrant expansion of CD11cdull CD8
+ non-plasmacytoid DCs in SOCS1-deficient mice
We observed that the lack of RAG, TCR
or CD28 in the SOCS1 KO background could rescue the lethality with acute inflammatory reaction that was seen in young SOCS1 KO mice. As shown in Fig. 1(A), SOCS1/CD28 DKO mice showed remarkable lifetime extension without any inflammatory symptoms until 3 month of age. However, after 3 months of age, most of these mice suffered various types of inflammatory lesions. We previously reported that the DCs from the SOCS1-deficient mice were highly activated, and these activated DCs were responsible for the development of inflammatory lesions at a later age (29).
We first observed DC sub-population in the spleen or thymus of young healthy mice (<10 week old) in the absence of SOCS1 protein. The CD11c+ cell number slightly increased in the SOCS1/CD28 DKO mice compared with B6 [wild-type (WT)] and CD28 KO mice (Fig. 3A). The proportion of CD8
+ DCs (CD8 DCs) in the SOCS1/CD28 DKO DCs was almost two times higher than those of normal B6 and CD28 KO mice (Fig. 1B). Similar increase in CD8 DC subsets was also observed in SOCS1/TCR DKO mice (Fig. 1B). A previous study reported that massive expansion of DCs in thymus was found in SOCS1 KO-Tg mice (29). Thus, we investigated whether the increased DCs in SOCS1-deficient thymus express CD8
. Similar to the splenic DCs, proportions of the CD8 DCs from thymus of SOCS1/CD28 DKO were clearly higher than those of B6 (WT) and CD28 KO mice (Fig. 1C). Therefore, in the absence of SOCS1 protein, the proportion of the CD8 DC subsets consistently increases in both spleen and thymus irrespective to the development of acute inflammatory response.
If the expression level of SOCS1 influences the development of the CD8 DC, CD8
+ and CD8
DC subsets may exhibit distinct expression profiles of SOCS1 mRNA. We compared the mRNA levels of SOCS1 and SOCS3 between freshly isolated CD8
+ and CD8
DC populations in normal B6 and CD28-deficient mice. Similar levels of SOCS3 expression were found in CD8
+ and CD8
DC subsets (Fig. 1D). However, the expression levels of SOCS1 mRNA were clearly distinctive in these two subsets. The CD8
DCs showed
200 times higher SOCS1 mRNA than those of CD8
+ DCs, suggesting that SOCS1 expression level may be critical in controlling the balance between CD8
+ and CD8
DC subsets.
Mouse splenic DCs can be divided into two major subsets on the basis of the expression levels of CD11c, CD11chigh and CD11cdull, which were defined as conventional DCs and plasmacytoid DCs, respectively. We examined that the expanded CD8 DCs in SOCS1/CD28 DKO mice could be classified as conventional or plasmacytoid DCs. The sorted CD11chigh or CD11cdull DC populations were stained with antibodies against CD8
, CD4 and CD11b (Fig. 2A). A marked increase of CD8
+ DCs was found in CD11cdull CD11b, suggesting that the expanded CD8 DCs correspond to plasmacytoid DCs. Conventional and plasmacytoid DCs are known to be defined as CD11chigh CD11b B220 CD45RA and CD11cdull CD11b B220+ CD45RA+, respectively. However, the CD11cdull CD11b CD8
+ DCs in SOCS1/CD28 DKO mice exhibited B220, and in CD11cdull population, CD45RA cells increased in SOCS1-deficient condition while CD45RA+ cells decreased (Fig. 2B and C) (32). Similar surface phenotype was also observed in DCs from SOCS1/TCR DKO (data not shown). These results indicate that the CD8 DCs increase in SOCS1-deficient mice could be classified as neither conventional nor plasmacytoid DCs, and this subset is quite minor in normal mice.

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Fig. 2. Expansion of unique CD8 DC subset in SOCS1-deficient mice. (A) CD11c+ DCs from CD28 KO or SOCS1/CD28 DKO mice were separated into CD11c high and low population. These CD11chigh and CD11cdull gated fractions were stained with the antibodies against either CD4 or CD11b versus CD8. (B) The increased CD11cdull CD8+ DCs were B220. CD11c+ DCs from CD28 KO or SOCS1/CD28 DKO mice were stained with the antibodies against B220 and CD11b. (C) The increased CD11cdull CD8+ DCs and CD45RAdull CD11c+ DCs from CD28 KO or SOCS1/CD28 DKO mice were stained with the antibodies against CD11c and CD45RA mAb, and the proportion of CD45RA dull and high were assessed in CD11cdull population.
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We further studied whether the proportional alteration observed in the absence of SOCS1 resulted from an increase in the absolute cell numbers of the CD8 DCs or a down-regulation of other DC subsets that are major in normal control mice. In spleens from SOCS1/CD28 DKO mice, the cell numbers of CD11chigh DCs slightly increased in comparison with normal B6 and CD28 KO mice, whereas the number of CD8 DCs in CD11cdull subsets from SOCS1/CD28 DKO mice was almost six times higher than that in CD28 KO mice (Fig. 3A), indicating that the abnormal CD8 DC subsets could drastically expand without a loss of other DC subsets in the SOCS1-deficient mice.
Histological localization of the abnormal CD8 DCs in splenic microenvironments was further examined by immunohistology. Frozen splenic sections from B6, CD28 KO and SOCS1/CD28 DKO mice were co-stained with CD11c in combination with CD4, CD8 or B220. The histological overall architecture in the spleens of SOCS1/CD28 DKO mice was indistinguishable from those in normal control C57BL/6 and CD28 KO mice as a clear distinction of white pulp, T cell zone and B cell follicles with hematoxylin staining (Fig. 3B). In normal C57BL/6 and CD28 KO mice, most CD8 single-positive cells were clearly discernible in the T cell area, while CD8 and CD11c double-positive cells were localized in marginal zone (33). However, appearance of the cells co-stained with CD11c and CD8 was more prominent in splenic sections of SOCS1/CD28 DKO mice. Co-localizations of CD11c and CD8 cells are detected deep within red pulp and T cell zone, outside of the B cell follicles and marginal zone, indicating that SOCS1/CD28 DKO mice exhibited relatively high number of CD8 DCs, and their distribution pattern was clearly distinct from CD8 DCs from normal C57BL/6 and CD28 KO mice.
CD8 DCs in SOCS1-deficient mice are responsible for BLyS/BAFF and IFN-
production
We previously described that the DCs developed in the absence of SOCS1 were highly activated and secreted B cell growth factor, BLyS/BAFF (29). To confirm that SOCS1/CD28 DKO DCs are activated even in the absence of any inflammatory symptoms, we analyzed BLyS/BAFF expressions in freshly isolated splenic DCs from CD28 KO and SOCS1/CD28 DKO mice. The CD8+ DCs from SOCS1/CD28 DKO spleen constitutively expressed higher levels of BLyS/BAFF mRNA than those of CD28 KO (Fig. 4A). Therefore, the abnormal CD8 DCs in SOCS1-deficient mice may be responsible for major source of BLyS/BAFF, which generates auto-antibody in SOCS1-deficient mice.
To further investigate the difference in functional features of the abnormal CD8 DCs, we characterized the sensitivity against pathogen-associated molecular patterns (PAMPs) by the assessment of the expressions of TLR4, TLR9 and CCR7 in freshly isolated splenic DCs from B6, CD28 KO and CD28/SOCS1 DKO mice. As shown in Fig. 4(B), SOCS1/CD28 DKO DCs showed higher TLR9 expression and slightly higher CCR7 expression in comparison with B6 and CD28 KO DCs, while there is slight reduction in the expressions of TLR4. Therefore, the SOCS1-deficient DCs may be more sensitive to prokaryotic DNA like CpG, rather than to LPS in the innate response.
The production of cytokines and chemokines related with inflammatory reaction was comprehensively analyzed in response to CpG. DCs from SOCS1/CD28 DKO mice exhibited higher production of many inflammatory cytokines, such as IFN-
, IL-3, IL-4, IL-6 and IL-12, than those of CD28 KO-derived DCs (Fig. 5A). In previous reports, we described that the macrophages and DCs developed in the absence of SOCS1 protein were highly sensitive to LPS stimulation (29, 34). We found that CpG stimulation also induced 10 times more IFN-
production (Fig. 5B) in comparison with LPS stimulation (data not shown). This difference may reflect the difference in the expression levels of TLR9 mRNA between CD28 KO and SOCS1/CD28 DKO DCs because the expression levels in SOCS1-deficient DCs were 2.5 times higher than those of control mice (Fig. 4B). Moreover, we examined whether the abnormal CD8 DC is responsible for the enhancement of IFN-
production in the absence of SOCS1. Splenic CD11c+ population was isolated from CD28 KO and SOCS1/CD28 DKO mice and was stimulated with CpG. The IFN-
-producing cells were measured by intracellular staining in conjunction with CD8 staining. As shown in Fig. 5(D), CD8+ DC derived from SOCS1/CD28 DKO mice was an IFN-
-producing population in response to CpG, indicating that high amount of IFN-
from SOCS1-deficient DCs resulted from CD8 DC expansion. Taken together, the abnormal CD8 DC subsets that expanded in the absence of SOCS1 protein have unique character of exhibiting massive IFN-
production in response to PAMPs.
Role of the abnormal CD8 DC subsets as immune modulator
Next, we examined whether the massive IFN-
production from abnormal CD8 DCs contribute to the Th1 dominancy in SOCS1-deficient mice. To examine a role of the SOCS1-deficient CD8 DC subsets in the antigen-specific reaction, DCs from CD28 KO and SOCS1/CD28 DKO were added into the co-culture of OT-II-derived T cells, BM-DC from B6 mice and OVA. After 5 days, primed T cells were re-stimulated with anti-TCR mAb. IFN-
- and IL-4-producing cells were analyzed by intracellular staining. Under this circumstance, OT-II-derived T cells shifted into Th1 cells (at proportional range between 24 and 32%), and the addition of DCs from CD28 KO mice slightly reduced the Th1 development at high dose. Unexpectedly, the addition of DCs from SOCS1/CD28 DKO markedly down-modulated Th1 development of OT-II-derived T cells (from 24 to 5%). Moreover, these suppressive effects were attenuated with the addition of anti-IFN-
mAb (Fig. 6), suggesting that the IFN-
production from CD8 DC subsets in SOCS1-deficient mice is rather harmful for the antigen-induced Th1 development.
SOCS1-deficient DCs fail to induce allogeneic MLR
Previous literatures accumulated evidence that CD11cdull population, including plasmacytoid DCs and CD45RBhigh tolerogenic DCs, down-regulated allo- and antigen-specific naive T cell response (35, 36). This motivated us to investigate whether the CD11cdull CD8 DC subsets expanded in SOCS1-deficient mice have similar regulatory function in allogeneic mixed lymphocyte reaction (MLR). The spleen-derived CD11c+ cells were purified from CD28 KO, SOCS1/CD28 DKO, TCR KO and SOCS1/TCR DKO mice with B6 background, and co-cultured with allogeneic CD4+ T cell from BALB/c for 48 h. DCs from both SOCS1/CD28 DKO and SOCS1/TCR DKO mice promoted much less allogeneic MLR in comparison with those in DCs from B6, CD28 KO and TCR KO mice (Fig. 7A). These results suggest that the SOCS1-deficient DCs that contain abnormal subsets of CD8 DCs were impaired in the function as APCs for allogeneic MLR.
Recently, correlation between IDO expression and immune suppression has been confirmed in several experimental systems (3739). cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig-treated DCs and macrophages down-modulate clonal expansion of T cells (40). Since the IDO expression is controlled by the IFN-
, we assume that IFN-
secreted from abnormal subsets of CD8 DCs induces a higher level of IDO expression in SOCS1-deficient DCs, which is highly sensitive to IFN-
compared with normal DCs. In order to examine this possibility, expressions of IDO mRNA in DCs from CD28 KO and SOCS1/CD28 DKO were analyzed in the presence or absence of CpG stimulation. DCs from SOCS1/CD28 DKO highly expressed IDO mRNA in comparison with those of CD28 KO (Fig. 7B), and the reduction of allo-MLR response was partially recovered by the addition of the pharmacological IDO-specific inhibitor 1-methyl-DL-tryptophan (Fig. 7C).
IL-10 is a critical cytokine to regulate the function and development of tolerogenic DCs. The activated tolerogenic CD8 DCs secretes IL-10, which controls development of Tr1 (36). Thus, we measured IL-10 production from CD28 KO and SOCS1/CD28 DKO DCs in the stimulation with CpG. SOCS1/CD28 DKO DCs secreted higher amounts of IL-10 in comparison with those of CD28 KO DCs (Fig. 7D). Taken together, IDO as well as IL-10 may be synergistically responsible for the impairment of SOCS1-deficient DCs in the induction of allo-MLR.
SOCS1-deficient DC has a defect in the expression of MHC class II and co-stimulatory molecules
The impairment in the induction of allo-MLR can also be explained by the defects in APC activity, such as the down-regulation of MHC and co-stimulatory molecules. Splenic DCs from CD28 KO and SOCS1/CD28 DKO mice were cultured for 24 h in the presence of GM-CSF and CpG, and the expression levels of MHC class II molecules, I-Ab and co-stimulatory molecules, CD40, CD80 and CD86 were then studied. DCs from SOCS1/CD28 DKO exhibited noticeable reduction of the CD11high DCs expressing high level of I-Ab (Fig. 7E). DCs from SOCS1/CD28 DKO expressed almost similar levels of CD40 and CD86 compared with those of CD28 KO in the absence of CpG stimulation. DCs from normal CD28 KO mice showed clear induction of CD40 and CD86 expressions in response to CpG. However, SOCS1/CD28 DKO DCs exhibited remarkably low expression levels of CD40 and CD86 molecules as compared with CD28 KO DCs (Fig. 7F). These results indicate that the expressions of MHC class II and co-stimulatory molecules are down-modulated in CD8 DCs that expand in SOCS1-deficient mice. Such defects may partly explain the impairment of SOCS1-deficient DCs in the induction of allo-MLR.
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Discussion
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The present study has demonstrated that the SOCS1 expression level is markedly high in freshly isolated conventional DCs that are major subsets in normal control mice, and the expression levels of SOCS1 during DC maturation may be critical in maintaining homeostasis of DC subsets. In the absence of SOCS1 protein, the numbers of minor subsets of DCs, CD11cdull CD8
+ CD11b CD45RA B220 that could be classified as neither conventional nor plasmacytoid DCs, were remarkably increased. This CD8 DC subset constitutively expressed BLyS/BAFF, and secreted a high-concentration IFN-
in response to PAMPs, resulting in aberrant auto-antibody production and Th1-dominant condition to facilitate inflammatory response. However, the CD8 DC subset in SOCS1-deficient mice not only expressed immune suppressive modulator, IDO, but also had a defect in the induction of class II and co-stimulatory molecules, thus this CD8 DC subset down-modulated the CD4+ T cell-mediated allogeneic and antigen-induced responses. These evidences indicate that the aberrant expansion of the CD8 DCs is not beneficial for a proper system in maintaining immune surveillance system. Therefore, we conclude that the SOCS1 expression levels during DC maturation process could be a key to maintain homeostasis of immune system.
SOCS1-deficient mice with acute inflammatory pathology die within 3 weeks of age. We previously demonstrated that the SOCS1 KO-Tg rescues the neonatal death, but develops systemic autoimmune-like symptoms with hypergammaglobulinemia and chronic inflammatory lesions (29). The life span was relatively extended in SOCS1/CD28 DKO and SOCS1/TCR
DKO mice. CD28 is a co-stimulatory molecule critical for primary T cell activation and IL-2 production, suggesting that the T cell is involved in the neonatal death of SOCS1-deficient mice. On the other hand, the doubly deficient mice developed chronic autoimmune-like inflammatory disease independent of the presence and activation of T cells. The production of anti-dsDNA antibody in sera seems to be controlled by the DC subset that secretes both IFN-
and BLyS/BAFF (29). Indeed, the CD8 DC subsets expanded in SOCS1-deficient mice constitutively express BLyS/BAFF. Therefore, the aberrant expansion of CD8 DCs may interpret the development of systemic autoimmune symptom in SOCS1-deficient mice.
SOCS1 is highly expressed in freshly isolated CD8 DC subsets, which exhibit either CD4+ CD8 or CD4 CD8 surface phenotype. In case of T and B cells, and macrophage, the expression levels of SOCS1 are quite low or even undetectable. Its expression levels are rapidly and drastically increased only when cells are stimulated with either cytokines or PAMPs. However, the SOCS1 expression levels in resting conventional DC subsets are >1000 times higher than those in T and B cells (data not shown). Interestingly, the expression levels of SOCS1 are different among DC subsets. Its expression levels in CD8+ DCs are
100 times lower than those in CD8 DCs that contained main subsets of conventional DCs. These results clearly address that the expression levels of SOCS1 have a significant role in maintaining the homeostasis among normal DC subsets.
The aberrant expansion of the CD8 DC subset was commonly observed in SOCS1-deficient strains with a CD28 KO and TCR
KO background. Previous literatures accumulated evidences to claim that the CD8
+ DC subsets revealed a unique feature as immune modulator. The spleen-derived CD11chigh lymphoid CD8
+ DC has been characterized such that these DCs are the major producer of IL-12 and prime for Th1, whereas it is also reported that CD8
+ DCs have tolerant function to down-modulate antigen-specific T cell proliferation via the induction of regulatory T cells (10, 41). Plasmacytoid DC also indicates a marked reduction of the naive T cell stimulatory potential with a low level of MHC and co-stimulatory molecules (42). Since CD8 DCs from SOCS1-deficient DCs from either CD28 or TCR
KO background reduce the expressions of co-stimulatory molecules, their functional feature resembles lymphoid CD8
+ DCs and plasmacytoid DCs. However, based on the cell-surface phenotype, the CD8 DC subsets exhibit CD11cdull B220. Moreover, histological geography is also different from conventional DCs. The abnormal CD8 DCs showed spread distributions in red pulp in the spleen, but in neither marginal zone nor T cell area of the periarteriolar lymphatic sheaths, where most of DC subsets are localized (33, 43). Therefore, the CD8 DC expanded in SOCS1-deficient mice was a unique minor subset in normal control mice. This subset distributes to not only the spleen but also lymph node and thymus in the absence of SOCS1.
The expanded CD8 DCs induced noticeable reduction of stimulatory potential in the allospecific T cell proliferation. This reduction may be partly contributed to the down-regulation of MHC class II and co-stimulatory molecules. The reduction of co-stimulatory molecules and alloresponse were completely recovered in SOCS1/IFN-
DKO mice (data not shown), suggesting that IFN-
secreted from abnormal CD8 DCs may cause such reduction. This result has suggested the possibility that the excess amount of IFN-
causes impairment in the stimulatory potential for the alloresponse. However, the administration of normal DCs with excess amount of IFN-
did not alter the expression of MHC class II and co-stimulatory molecules, indicating that IFN-
may act on distinct pathway.
Since DCs from SOCS1/CD28 DKO act as down-modulator for the OVA-induced Th induction in OT-II Tg T cells, it is thought that the reduction of CD4+ T cell-mediated allo-MLR also contributes to the regulatory effect of the SOCS1 KO-derived DCs, rather than to the defect of the antigen-presenting ability. DCs expressing CD8
, which were already described (10, 44), promote tolerance to peripheral self-antigens and tumor immunity. A recent literature demonstrated that CTLA4-Ig treatment in vivo induced tolerogenic DCs expressing IDO, and CD11c+ B220 CD8
+ non-plasmacytoide DCs expressed high levels of IDO (9, 40). The tolerogenic potential with SOCS1/CD28 DKO-derived DCs seems to correlate with high IDO expression levels and the aberrant expansion of CD8 DCs, which showed identical surface phenotype to the CTLA4-Ig-induced tolerogenic DCs. Since IDO expression is controlled by IFN-
(44), the high IDO expression in SOCS1-deficient DCs is consistent with the fact that such DCs produce higher amount of IFN-
and exhibit higher sensitivity against IFN-
. Moreover, treatment with pharmacological IDO inhibitor partly canceled the reduction of CD4+ T cell-mediated allo-MLR, suggesting that the CD8 DCs expanded in the SOCS1-deficient environment has a potential to act as tolerogenic DCs by induction of IDO. Antigen-pulsed CD11cdull CD45RB+ CD8
+ DCs specifically control the differentiation of Tr1, which shows tolerogenic functions through IL-10 production (36). The expanded CD8 DC in SOCS1-deficient mice is also CD11cdull CD45RB+ (data not shown), thus it is possible that CD8 DCs may control the regulatory function via the involvement of Tr1. However, we did not have evidence to confirm the Tr1 involvement. This remains to be further examined.
Finally, the abnormal CD8 DC subset may be harmful in maintaining a proper immune surveillance system because the presence of CD8 DC results in down-regulation of allo- and antigen-specific immune responses and Th1 skewing. Therefore, high expression of SOCS1 during DC maturation is a critical process to inhibit the aberrant expansion of CD8 DC subset.
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Acknowledgements
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Jun Tsukada is research fellow of Junior Research Associate in Riken. This work was supported by Grants-in-Aid for Scientific Research on Priority area from the Ministry of Education, Culture, Sports, Science, and Technology (Japan).
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Abbreviations
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---|
APC | antigen-presenting cell |
APRIL | a proliferation-inducing ligand |
BLyS/ BAFF | B lymphocyte stimulator/B cell activation factor of the tumor necrosis factor family |
BM-DC | bone marrow-derived dendritic cell |
CTLA4 | cytotoxic T lymphocyte antigen 4 |
DC | dendritic cell |
GM-CSF | granulocyte macrophage colony-stimulating factor |
[3H]TdR | [3H]thymidine |
IDO | indoleamine 2,3-dioxygenase |
JAK | Janus kinase |
MLR | mixed lymphocyte reaction |
OVA | ovalbumin peptide |
PAMP | pathogen-associated molecular pattern |
RAG2 | recombination activating gene 2 |
SOCS | suppressor of cytokine signaling |
SOCS1 KO-Tg | SOCS1-transgenic mice on a SOCS1 KO background |
STAT | signal transduction and activators of transcription |
TLR | Toll-like receptor |
WT | wild type |
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Notes
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Transmitting editor: T. Watanabe
Received 15 March 2005,
accepted 13 June 2005.
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