Skip to main content
NCBI home page
Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 2015 May 14;181(1):76–86. doi: 10.1111/cei.12629

Interleukin-2 treatment reverses effects of cAMP-responsive element modulator α-over-expressing T cells in autoimmune-prone mice

K Ohl *,†,1, A Wiener *,1, A Schippers *, N Wagner *, K Tenbrock *,✉
PMCID: PMC4469157  PMID: 25817470

Abstract

Systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), are often characterized by a failure of self-tolerance and result in an uncontrolled activation of B cells and effector T cells. Interleukin (IL)-2 critically maintains homeostasis of regulatory T cells (Treg) and effector T cells in the periphery. Previously, we identified the cAMP-responsive element modulator α (CREMα) as a major factor responsible for decreased IL-2 production in T cells from SLE patients. Additionally, using a transgenic mouse that specifically over-expresses CREMα in T cells (CD2CREMαtg), we provided in-vivo evidence that CREMα indeed suppresses IL-2 production. To analyse the effects of CREMα in an autoimmune prone mouse model we introduced a Fas mutation in the CD2CREMαtg mice (FVB/Fas–/–CD2CREMαtg). Overexpression of CREMα strongly accelerated the lymphadenopathy and splenomegaly in the FVB/Fas–/– mice. This was accompanied by a massive expansion of double-negative (DN) T cells, enhanced numbers of interferon (IFN)-γ-producing T cells and reduced percentages of Tregs. Treatment of FVB/Fas–/–CD2CREMαtg mice with IL-2 restored the percentage of Tregs and reversed increased IFN-γ production, but did not affect the number of DNTs. Our data indicate that CREMα contributes to the failure of tolerance in SLE by favouring effector T cells and decreasing regulatory T cells, partially mediated by repression of IL-2 in vivo.

Keywords: CREM, IL-2, regulatory T cells, SLE

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disorder that affects nearly all organ systems in the human body. SLE can manifest in genetically susceptible individuals and is triggered by environmental and hormonal factors 1,2. It is characterized by a failure of immunological self-tolerance to ubiquitous nuclear antigens and results in an uncontrolled activation of self-reactive B and T cells. 3. T lymphocytes from patients with SLE display a complex array of cellular, molecular and signalling anomalies, many of which have been attributed to increased expression of the transcriptional regulator, cAMP-responsive element modulator α (CREMα) 4.

CREMα belongs to the superfamily of bZip proteins. Members of this family of proteins contain a basic domain/leucine zipper domain, which binds to an 8-base pair palindromic DNA sequence (cAMP response element, TGACGTCA) 5–7. We have shown recently that transgenic over-expression of CREMα in T cells results in the down-regulation of interleukin (IL)-2 transcription and enhancement of a T helper type 17 (Th17)-type immune response 8. This was confirmed in a model of lipopolysaccharide (LPS)-induced acute lung injury, where mice with T cell-specific over-expression of CREMα displayed severely enhanced disease activity accompanied by decreased expression of IL-2, enhanced expression of IL-17 and decreased expression of forkhead box protein 3 (FoxP3), which is the lineage-specific transcription factor of regulatory T cells (Treg) 9.

CD4+CD25+FoxP3+ Treg cells inhibit activation and expansion of CD4+ helper T cells, but also counteract differentiation of cytotoxic CD8+ T cells and B cell activation 10–13, and therefore represent an important mechanism to control peripheral tolerance. Complete loss of FoxP3 protein and a subsequent lack of Treg cells have fatal effects. FoxP3-deficient scurfy mice develop signs of autoimmune inflammation with exaggerated Th1, Th2 and Th17 responses and eventually die by 3–4 weeks of age 14,15. Reports on mutations in the FoxP3 gene in humans show associations with a severely impaired immune regulation, polyendocrinopathy, enteropathy and X-linked syndrome 16,17.

Reports on the numbers and function of Treg cells in SLE and murine lupus models are contradictory. Surprisingly, recent papers have demonstrated normal numbers of fully functional Treg cells in the peripheral blood of SLE patients 18,19. Nevertheless, considering the high numbers of activated inflammatory T cells, a disturbed balance between effector T cells and Treg cells is evident in SLE and in murine lupus models, which becomes aggravated with disease progression 20–22. Adoptive transfer experiments with Treg cells resulted in a delayed onset of disease and reduced mortality of lupus-prone mice 21,23,24, which demonstrates that Treg cells are indeed beneficial in SLE. Current strategies to maintain Treg cell homeostasis in SLE are therefore promising. IL-2 is critically required for the survival and competitive fitness of Treg cells in vivo 25–27. It is therefore supposed that IL-2 deficiency leads to the disruption of the homeostasis between Treg cells and effector T cells in SLE. Treatment of lupus-prone mice with IL-2 re-established the homeostatic proportion of Treg cells to T effector cells and delayed disease progression in (NZB×NZW) F1 mice as well as in Murphy Roths Large lymphoproliferation (MRL/lpr) mice 21,28, both of which are established murine lupus models. Conversely, reduction of Treg cells in young clinically healthy (NZB×NZW) F1 animals by IL-2 neutralization or CD25 depletion accelerated the onset of autoimmune disease 21,29.

Through a co-operative analysis of CREMα-over-expressing Fas–/– mice [Friend leukaemia virus, strain B (FVB)/Fas–/–CD2CREMαtg] and Fas–/– mice (FVB/Fas–/–) we aimed to evaluate the importance of CREMα for the homeostatic balance of effector T cells and Treg cells in a lupus-like model. Secondly, through IL-2 treatment, we aimed to analyse the contributions of IL-2 to this process.

Materials and methods

Animals

Experiments were performed with FVB wild-type, FVB/Fas–/– and FVB/Fas–/–CD2CREMαtg mice 8. The study was approved by the regional government authorities and animal procedures were performed according to German legislation for animal protection. Permission for the projects was granted by the Regierungspräsident/LANUV Nordrhein Westfalen (AZ 87-51.04.2010.A327).

IL-2 treatment

For long-term IL-2 treatment, FVB/fas–/– and FVB/fas–/–CD2CREMαtg mice were injected intraperitoneally (i.p.) with a high dose of 25 ng/g body weight recombinant murine IL-2 (rmIL-2) (R&D Systems, Minneapolis, MN, USA) in 200 µl sterile phosphate-buffered saline (PBS) every 12 h within 24 h (three times in total; short-term treatment). The treatment was repeated every 5 days with 25 ng/g body weight of rmIL-2 for a total of five times after the initial short-term treatment (leaned on 21). In an equivalent long-term experiment mice were injected i.p. with a low dose of 25 ng per animal rmIL-2. Age- and gender-matched control and IL-2-treated animals were killed 24 h after the last injection. Serum and tissue were harvested for analysis.

Assessment of lymphadenopathy

Blinded scoring of lymphadenopathy in control and IL-2-treated FVB/Fas–/– and FVB/Fas–/–CD2CREMαtg mice was performed by three observers on a 0–4+ scale, as described previously 30, and scored as follows: 0 = no detectable lymphadenopathy; 1+ = mild submandibular adenopathy only; 2+ = moderate submandibular adenopathy only; 3+ = submandibular adenopathy plus one other palpable node; and 4+ = diffuse lymphadenopathy.

Life expectancy

Kaplan–Meier survival curves were used to assess the life expectancy of FVB/Fas–/– mice compared to FVB/Fas–/–CD2CREMαtg mice with GraphPad Prism software (GraphPad, San Diego, CA, USA). Eighty-seven FVB/Fas–/– and 160 FVB/Fas–/–CD2CREMαtg mice were examined. For statistical analysis the log-rank (Mantel–Cox) test was performed.

Flow cytometric analysis

For surface staining, single-cell suspensions were prepared from spleens and lymph nodes and stained with the following specific antibodies: anti-CD3-allophycocyanin (APC) (eBioscience, San Diego, CA, USA), anti-CD4-fluorescein isothiocyanate (FITC) (eBioscience), anti-CD8-Pacific Blue (eBioscience), anti-CD69-phycoerythrin (PE) (eBioscience), anti-CD3-PE (eBioscience), anti-CD25-PE (eBioscience), anti-CD19-FITC (eBioscience), anti-CD11b-PB (eBioscience) and anti-CD14-FITC (eBioscience). Murine anti-CREM antibody (Santa Cruz Technology, Santa Cruz, CA, USA) was labelled with Alexa-488 using the Invitrogen (Carlsbad, CA, USA) Alexa 488 labelling lit. Intracellular staining of FoxP3 and CREM was performed using the eBioscience FoxP3 staining buffer kit, according to the manufacturer's instructions. For measurement of intracellular interferon (IFN)-γ, IL-17a and IL-2 production, cells were treated with phorbol myristate acetate (PMA) (20 nM), ionomycin (2 μM) (both Sigma-Aldrich, St Louis, MO, USA) and GolgiPlug (BD Bioscience, Heidelberg, Germany) for 5 h and intracellular staining was performed with anti-IFN-γ-Alexa 647, anti-IL-17a-Alexa 488 and anti-IL-2-FITC (eBioscience). Samples were analysed using a fluorescence activated cell sorter (FACS)Canto II (BD) and data were analysed using FCS Express (De Novo Software, Glendale, CA, USA) software.

Enzyme-linked immunosorbent assay (ELISA)

Total immunoglobulin (Ig)G was measured in sera from control and IL-2-treated FVB/Fas–/–and FVB/Fas–/–CD2CREMαtg mice using the Ready-Set-Go ELISA system (Affymetrix; eBioscience, USA).

Reverse transcription–polymerase chain reaction (RT–PCR)

Total RNA from cells was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was then generated using the First-Strand cDNA Synthesis Kit (Fermentas, Baden-Württemberg, Germany), according to the manufacturer's instructions. Standard real-time PCR was carried out on TaqMan 7900 (Applied Biosystems/Life Technologies, Darmstadt, Germany) using the DNA intercalating dye SYBR Green. We used the following primer sequences: for β-actin, GAC TAC CTC ATG AAG ATC CTC ACC (forward) and TCT CCT TAA TGT CAC GCA CGA TT (reverse); for IL-2, gac ctc tgc ggc atg ttc tg (forward) and tca tcg aat tgg cac tca aat g (reverse); for inducible cAMP early repressor (ICER), ATG GCT GTA ACT GGA GAT GAA (forward) and AGC AAA TGT CTT TCA AAC TTT CAA (reverse). Relative quantification method was applied and delta Ct (ΔCt) values were determined by subtracting the Ct of the housekeeping gene from the Ct of the target gene for each sample, respectively.

Histochemical analysis of spleens

Spleens were harvested and frozen immediately in embedding media (Tissue-Tek; Sakura, Zoeterwoude, the Netherlands). Sections of 7 µm thickness were cut. CD3 were detected using a CD3e-APC antibody (eBioscience). Images were captured using a Cool SNAP HQ2 camera and an Axioplan 2 Imaging microscope (Zeiss, Oberkochen, Germany) with VisiView software.

Results

T cell-specific over-expression of CREMα enhances disease activity in Fas–/– mice

CREM is expressed at increased levels in T cells from SLE patients and lupus-prone MRL/lpr mice 31,32. A single mutation of the Fas gene (CD95) causes a spontaneous age-dependent lupus-like disease in susceptible mice from the MRL strain 33. To further analyse the effects of T cellular CREM expression in lupus we introduced a genetic deletion of CD95 (Fas−/−) into the CD2CREMαtg mice (FVB/Fas–/–CREMαtg mice), which over-express CREMα in T cells, and wild-type FVB/Fas–/– mice. The enhanced expression of CREMα in CD2CREMαtg and FVB/Fas–/–CREMαtg mice 8 (Supporting information, Fig. S1a) compared to FVB/wt and FVB/Fas–/–mice did not involve enhanced levels of ICER (inducible cAMP early repressor), an inducible isoform of CREM (Supporting information, Fig. S1b,c). FVB/Fas–/– mice already expressed increased levels of CREMα protein (P = 0·0184, Fig. 1a, Supporting information, Fig. S1a) in T cells and also developed a lymphadenopathy which proceeded with age and splenomegaly when compared to wild-type mice and (Fig. 1b,c). When compared to FVB/Fas–/–mice, FVB/Fas–/–CREMαtg mice suffered from massively enhanced lymphadenopathy 8 (Fig. 1b, 5–8-week-old mice, P < 0·001, 9–12-week-old mice, P < 0·001, 15–18-week-old mice, P < 0·01) and splenomegaly (Fig. 1c,d, P = 0·0076).

Figure 1.

Figure 1

Open in a new tab

Over-expression of cAMP-responsive element modulator α (CREMα) enhances lymphadenopathy and splenomegaly in a murine lupus model. (a) The frequency of CREMhighCD3+ cells in peripheral lymph nodes of three Friend leukaemia virus, strain B (FVB)/wild-type (WT) and three FVB/Fas–/– mice was quantified by flow cytometry. Data show mean percentage ± standard error of the mean (s.e.m.) of CREM+ cells in splenic CD3+ cells of FVB/WT and FVB/Fas–/– mice (two-tailed, unpaired t-test, P = 0·0184). (b) Assessment of lymphadenopathy in differently aged FVB/Fas–/– and FVB/Fas–/–CREMαtg mice. Data indicate mean ± s.e.m. of at least three mice per group (two-way analysis of variance, Bonferroni post-test, ***P < 0·001, **P < 0·001). (c) Representative image of spleens from 14-week-old WT (left), FVB/Fas–/– (in the middle) and FVB/Fas–/–CREMαtg (right) mice. (d) Weight of spleens from 10–11-week-old FVB/Fas–/– and FVB/Fas–/–CREMαtg mice. Data indicate mean ± s.e.m. of seven mice per group (two-tailed, unpaired t-test, P = 0·0076).

FVB/Fas–/–CREMαtg mice reveal increased inflammatory but decreased Treg cells

We next characterized the cells that caused the expansion of lymphoid tissue. In MRL/lpr mice, CD3+CD4−CD8− double-negative T cells (DNTs) account for the severe lymphadenopathy and splenomegaly 34. DNTs are expanded in the peripheral blood of SLE patients, can produce IL-17A, induce IgG and anti-DNA antibody production and contribute to tissue damage by infiltrating kidneys of SLE patients 35,36. FVB/Fas–/–mice also exhibited enhanced frequencies of DNTs in spleens and lymph nodes when compared to non-autoimmune wild-type mice (Fig. 2a, ***P = 0·0002, Fig. 2b). Interestingly, FVB/Fas–/–CREMαtg mice displayed greatly increased percentages of DNTs in comparison to wild-type mice, but also when compared to FVB/Fas–/– mice in spleens (Fig. 2a, ****P < 0·0001) and lymph nodes (Fig. 2b). With regard to absolute cell numbers, FVB/Fas–/–CREMαtg mice also displayed enhanced numbers of CD4+ T cells (P < 0·0001) and CD8+ T cells (P = 0·0003) and a massive expansion of DNT cells in spleen (Fig. 2c, P = 0·0011). Absolute numbers of macrophages (CD11b+ F4/80+), dendritic cells (CD11c+) and B cells (CD19+) cells were also higher in spleens of FVB/Fas–/–CREMαtg mice compared to FVB/Fas–/– mice (Fig. 2d). Nevertheless, percentages were reduced (Fig. 2e), due most probably to the massive expansion of DNTs. Immune-histological evaluation of spleen further demonstrated a dominant appearance of T cells (Fig. 2f). This suggests that DNT cells, which are already known to account for severe lymphadenopathy and splenomegaly in Fas–/– mice, also account for the further increased lymphadenopathy and splenomegaly in FVB/Fas–/–CREMαtg mice. Our observation thereby confirms recently published data in vivo, which demonstrate that CREMα contributes to generation of CD3+CD4−CD8− cells by repressing CD8 transcription 37. In addition to the high occurrence of DNTs, FVB/Fas–/–CREMαtg mice displayed a higher percentage of IFN-γ− and IL-17a-producing T cells in spleens when compared to FVB/Fas–/– mice (Fig. 3a,b). IL-21 levels were quite low, and did not differ between both groups (Fig. 3a). IFN-γ was produced predominantly by CD8+ T cells and CD4+ T cells and FVB/Fas–/–CREMαtg mice revealed enhanced percentages of IFN-γ-producing cells in both CD8+ and CD4+ T cells (Supporting information, Fig. S2a). DNTs rarely produced IFN-γ in FVB/Fas–/– and FVB/Fas–/–CREMαtg mice and levels were not significantly different between both groups (Supporting information, Fig. S2a). IL-17a was produced mainly by DNTs, but also by CD4+ and CD8+ T cells in FVB/Fas–/– mice and in all three T cells subsets to a higher extent in FVB/Fas–/–CREMαtg mice (Supporting information, Fig. S2b). This suggests that over-expression of CREMα does not affect a specific T cell subset but more amplifies inflammatory cytokine production of all observed T cell subsets in FVB/Fas–/– mice. IFN-γ as well as IL-17a appear to be involved critically in lupus pathogenesis. IFN-γ-deficient MRL/lpr mice are prevented from early death, as well as having reduced lymphadenopathy and reduced glomerulonephritis 38. IL-17-deficient mice are protected from development of lupus autoantibodies and glomerulonephritis in a pristine-induced lupus model 39. In contrast, compared to FVB/Fas–/– mice, the percentages of suppressive FoxP3+CD25+ cells in the splenic CD4+ population of FVB/Fas–/–CREMαtg mice were lower (Fig. 3c, P = 0·0370, Fig. 3d). Interestingly IL-2 expression, which we showed to be reduced in CD2CREMαtg mice 8 (Supporting information, Fig. S2c), was also reduced in FVB/Fas–/–CREMαtg compared to FVB/Fas–/– mice (Fig. 3e, P = 0·0377, Supporting information, Fig. S2c). These results indicate that CREMα over-expressing FVB/Fas–/– mice display higher percentages of IFN-γ− and IL-17a-producing inflammatory T cells while suppressive regulatory T cells and IL-2 expression are reduced.

Figure 2.

Figure 2

Open in a new tab

Enhanced splenomegaly and lymphadenopathy of mice are caused by a massive expansion of double-negative T cells (DNTs). Cells were isolated from spleen and cervical lymph nodes and flow cytometric analyses were performed. (a) Percentages of CD4−CD8− cells in splenic CD3+ T cells from Friend leukaemia virus, strain B/wild-type (FVB/WT) (circle dots), FVB/Fas–/– (squares) and FVB/Fas–/–cAMP-responsive element modulator (CREM)αtg (triangles) mice assessed by flow cytometry. Horizontal lines show mean ± standard error of the mean (s.e.m.). Each point represents data from one mouse (two-tailed, unpaired t-test, ***P = 0·0002, ****P < 0·0001). (b) Representative flow cytometry dot-plots gated on the CD3+ population in cervical lymph nodes of 7–8-week-old FVB/wild-type (WT) (left), FVB/Fas–/– (middle) and FVB/Fas–/–CREMαtg mice (right). DNT cells fill the lower left quadrant. (c) Absolute number of CD3+CD4+, CD3+CD8+ and CD3+CD4−CD8− cells in spleens of 12-week-old FVB/WT, FVB/Fas–/– mice and FVB/Fas–/–CREMαtg mice. Bars indicate means ± standard error of the mean (s.e.m.) of five mice per group (two-tailed, unpaired t-tests, CD3+CD4+ cells: P < 0·0001, CD3+CD8+ cells: P = 0·0003, CD3+CD4−CD8− cells: P = 0·0011). (d) Absolute numbers of CD11b+F4/80+, CD11c+ and CD19+ cells in spleens of 12-week-old FVB/Fas–/– mice and FVB/Fas–/–CREMαtg mice. Bars indicate mean ± s.e.m. of five mice per group (two-tailed, unpaired t-tests, CD11b+F4/80+ cells: P = 0·0005, CD11c+ cells: P = 0·0003, CD19+ cells: P = 0·0004). (e) Percentages of CD11b+F4/80+, CD11c+ and CD19+ cells in spleens of FVB/Fas–/– (squares) and FVB/Fas–/–CREMαtg (triangles) mice as assessed by flow cytometry. Horizontal lines show mean ± s.e.m. Each point represents data from one mouse (two-tailed, unpaired t-tests, CD11b+F4/80+ cells: P = 0·0005, CD11c+ cells: P = 0·0002, CD19+ cells: P < 0·0001). (f) Immunofluorescence of frozen spleen sections. The left panel shows a representative CD3 (red) staining in spleen of FVB/Fas–/– mice, the right panel shows a representative spleen section of FVB/Fas–/–CREMαtg.

Figure 3.

Figure 3

Open in a new tab

Friend leukaemia virus, strain B (FVB)/Fas–/–cAMP-responsive element modulator α (CREM)αtg mice exhibit higher numbers of activated T cells and interferon (IFN)-γ-producing T cells while regulatory T cells are reduced. Cells were isolated from spleen and cervical lymph nodes and flow cytometric analyses performed. (a) Percentage of CD3+IFN-γ+ cells, CD3+interleukin (IL)-17a+ and CD3+IL-21+ cells. Horizontal lines show means ± s.e.m. Each point represents data from one mouse (CD3+IFN-γ+ two-tailed, unpaired t-test, P < 0·0001, CD3+IL-17a+ one-tailed, paired t-test, P = 0·04). (b) Representative flow cytometry dot-plots show co-staining for CD3 and IFN-γ and for CD3 and IL-17a. (c) Percentage of CD25+forkhead box protein 3 (FoxP3)+CD4+ cells assessed by flow cytometry. Horizontal lines show means ± s.e.m. Each point represents data from one mouse (two-tailed, unpaired t-test, P = 0·0370). (d) Representative dot-plots gated on the CD4+ cell population show percentages of CD25+FoxP3+ cells. (e) N-fold IL-2 mRNA expression in splenic T cells from three FVB/Fas–/– CREMαtg mice compared to three FVB/Fas–/– mice (one-sample test, P = 0·0377).

T cell-specific over-expression of CREMα in Fas–/– mice enhances IgG production and reduces lifespan

We have shown previously that over-expression of CREMα in T cells resulted not only in T cell aberrancies, but also enhanced anti-ds DNA antibody production 8. We therefore analysed total IgG levels in sera and found enhanced IgG levels in FVB/Fas–/–CREMαtg mice compared to FVB/Fas–/– mice (P = 0·0218, Fig. 4a). Furthermore, FVB/Fas–/–CREMαtg mice exhibited a reduced lifespan (P = 0·0036, Fig. 4b). These results indicate that CREMα expression in T cells also has systemic effects in an autoimmune prone model. In summary, these data indicate that over-expression of CREMα aggravates the imbalance of Treg cells and inflammatory T cells in Fas–/– mice by enhancing numbers of DNTs and IFN-γ producing T cells and reducing frequencies of regulatory T cells. We have shown previously that CREMα critically influences IL-2 expression 8, and IL-2 expression is indeed reduced in FVB/Fas–/–CREMαtg (Fig. 3d). We therefore aimed to analyse if treatment of FVB/Fas–/–CREMαtg mice with IL-2 re-establishes T cell aberrancies.

Figure 4.

Figure 4

Open in a new tab

FVB/Fas–/– cAMP-responsive element modulator α (CREM)αtg mice display higher immunoglobulin (Ig)G levels in sera and suffer from a reduced lifespan. (a) IgG enzyme-linked immunosorbent assay (ELISA) of sera from 10–11-week-old mice. Bars indicate mean values of µg/ml IgG in serum ± standard error of the mean (s.e.m.) from three FVB/Fas–/– and four FVB/Fas–/–CREMαtg mice (two-tailed, unpaired t-test, P = 0·0218). (b) Life expectancy of FVB/Fas–/– and FVB/Fas–/– CREMαtg animals; 87 FVB/Fas–/– mice and 160 Fas–/– FVB/Fas–/–CREMαtg mice were observed (survival curve comparison, log-rank (Mantel–Cox) test, P = 0·0036).

High-dose IL-2 restores numbers of Treg cells in FVB/Fas–/–CREMαtg mice

Mice were treated with 25 ng/g body weight recombinant murine IL-2, as described previously 21. Administration of IL-2 reduced percentages of CD3+IFN-γ+ in the spleen of FVB/Fas–/–CREMαtg mice but not in FVB/Fas–/– mice (**P = 0·0023, Fig. 5a). In addition, percentages of regulatory T cells were increased in IL-2 treated FVB/Fas–/–CREMαtg mice (**P = 0·005, Fig. 5b). These results suggest that treatment of FVB/Fas–/–CREMαtg mice with IL-2 raises frequencies of suppressive Treg cells, while reducing frequencies of inflammatory IFN-γ-producing T cells, resulting in an improved homeostasis between Treg cells and inflammatory effector T cells (Fig. 5c). However, percentages of IL-17a-producing cells were not reduced by IL-2 treatment (data not shown). This can be explained by the fact that DNTs, which we identified as major IL-17 source, were not altered in FVB/Fas–/–CREMαtg and FVB/Fas–/– mice after IL-2 treatment (Fig. 5d). Consequently, splenomegaly and lymphadenopathy were also not affected (Fig. 5e,f). IL-2-treated FVB/Fas–/–CREMαtg mice exhibited a trend towards lower IgG levels in sera compared to untreated littermates; however, this did not reach statistical significance (Fig. 5g).

Figure 5.

Figure 5

Open in a new tab

High-dose interleukin (IL)-2 treatment of cAMP-responsive element modulator (CREM)αtg lupus-prone mice in vivo enhances numbers of regulatory T cells (Treg) cells, reduces percentages of interferon (IFN)-γ-producing cells but does not ameliorate splenomegaly or lymphadenopathy. Mice were either left untreated or treated with IL-2 25 ng/g every 5 days for 4 weeks. Cells were isolated from spleens of untreated Friend leukaemia virus, strain B (FVB)/Fas–/– mice (filled squares), IL-2 treated FVB/Fas–/– mice (open squares), untreated FVB/Fas–/–CREMαtg mice (filled triangles) and IL-2 treated FVB/Fas–/–CREMαtg mice (filled triangles) and flow cytometric analyses performed. Horizontal lines show means ± standard error of the mean (s.e.m.). Each point represents data from one mouse (a) Percentages of CD3+IFN-γ+ cells assessed by flow cytometry (two-tailed, unpaired t-tests, **P = 0·0023). (b) Percentages of CD25+forkhead box protein 3 (FoxP3)+CD4+ cells (two-tailed, unpaired t-tests, *P = 0·0145). (c) Ratio between CD25+FoxP3+CD4+ cells and IFN-γ+ Τ cells (two-tailed, paired t-test, **P = 0·0016). (d) Percentages of CD3+CD4−CD8− cells. (e) Weight of spleens. Bars indicate mean ± s.e.m. of four to five mice per group. (f) Assessment of lymphadenopathy. Bars indicate mean ± s.e.m. of nine to 11 mice per group. (g) Immunoglobulin (Ig)G enzyme-linked immunosorbent assay (ELISA) of sera from 10–11-week-old mice. Data indicate mean values of µg/ml IgG in serum ± s.e.m. from four FVB/Fas–/– and four FVB/Fas–/–CREMαtg mice.

Low-dose IL-2 restores Treg cells and reverses enhanced IgG levels in FVB/Fas–/–CREMαtg mice

As IL-2 can also enhance immune responses by promoting the proliferation and generation of effector T cells, and low doses of rIL-2–IL-2 monoclonal antibody (mAb) complexes stimulate Treg cells but not memory/effector T cells preferentially 40, we aimed to analyse if low doses of IL-2 are sufficient to increase numbers of Treg cells. To this end, we treated both strains with 25 ng/mouse of rmIL-2 every 5 days for 4 weeks, as described previously. Again, we observed neither a reduction of splenomegaly (Fig. 6a) lymphadenopathy and (Fig. 6b) nor reduced frequencies of DNT cells (Fig. 6c) in FVB/Fas–/–CREMαtg or FVB/Fas–/– mice after IL-2 administration. However, even low doses of IL-2 were sufficient to increase Treg cells in FVB/Fas–/–CREMαtg mice, while not in FVB/Fas–/– mice (P = 0·0145, Fig. 6d), and to reduce splenic CD3+IFN-γ+ T cells of FVB/Fas–/–CREMαtg mice (P = 0·0177, Fig. 6e). These results indicate that even low doses of IL-2 improve Treg cell/effector T cell homeostasis in FVB/Fas–/–CREMαtg mice. Interestingly, this dose also significantly reduced IgG levels in sera of FVB/Fas–/–CREMαtg mice (P = 0·0173, Fig. 6f). From this we conclude that low-dose IL-2 treatment of FVB/Fas–/–CREMαtg mice not only affects T cells, but also reverses enhanced IgG production of plasma cells.

Figure 6.

Figure 6

Open in a new tab

Low-dose interleukin (IL)-2 treatment of Friend leukaemia virus, strain B (FVB)/Fas–/–cAMP-responsive element modulator (CREM)αtg mice in vivo reduces interferon (IFN)-γ+ cells, amplifies regulatory T cells (Treg) cells and reduces IgG sera antibodies but has no effect on lymphadenopathy, splenomegaly or double-negative cell (DNT) expression. Mice were left untreated or treated with 25 ng/mice IL-2 every 5 days for 4 weeks. (a) Weight of spleens from three to five animals. Horizontal bars indicate mean ± s.e.m. (b) Assessment of lymphadenopathy. Bars indicate mean ± s.e.m. from three untreated FVB/Fas–/– mice, five IL-2 treated FVB/Fas–/– mice, seven FVB/Fas–/–CREMαtg and 10 IL-2 treated FVB/Fas–/–CREMαtg mice (two-tailed, unpaired t-tests, *P = 0·0337). (c–e) Cells were isolated from spleens of untreated FVB/Fas–/– mice (filled squares), IL-2-treated FVB/Fas–/– mice (open squares), untreated FVB/Fas–/–CREMαtg mice (filled triangles) and IL-2-treated FVB/Fas–/–CREMαtg mice (filled triangles) and flow cytometric analyses were performed. Horizontal lines indicate mean ± s.e.m. Each point represents data from one mouse (two-tailed, unpaired t-tests, *P < 0·05, **P < 0·01. (c) Percentages of CD3+CD4−CD8− cells. (d) Percentages of CD25+forkhead box protein 3 (FoxP3)+CD4+ cells. (e) Percentages of CD3+IFN-γ+ cells. (f) Immunoglobulin (Ig)G enzyme-linked immunosorbent assay (ELISA) of sera from 10–11-week-old mice. Data indicate mean values of µg/ml IgG in serum ± s.e.m. (paired, two-tailed t-test, P = 0·0173).

Discussion

The critical balance of effector T cells and Treg cells seems to be a central pathogenic mechanism in human SLE. T cells from SLE patients exhibit defective IL-2 production and similarly defective IL-2 production was observed in murine lupus models 21,41–45. Among other factors, the molecular mechanism underlying the IL-2 defect in SLE is explained by CREMα over-expression 46,47. CREMα is over-expressed in SLE T cells and CREMα promoter activity correlates with disease activity in these individuals 48. Anti-CD-3 autoantibodies in sera of SLE patients mediate a translocation of calcium calmodulin kinase IV to the nucleus, and subsequently enhance transcription and expression of CREMα, which further enhances CREMα binding to the IL2 promoter and decreases IL-2 production 49. As found in human CREMα-over-expressing SLE T cells, CD2CREMαtg and FVB/Fas–/–CREMαtg murine T cells produce less IL-2 compared to their littermates 8 and, conversely, IL-2 levels are increased in CREM–/– mice compared to wild-type mice (Verjans et al., submitted). Here we show that transgenic over-expression of CREMα in T cells results in a severely amplified disease in a Fas–/– lupus-like mouse model with a massively expanded DNT pool and increased numbers of IFN-γ-producing T cells, on one hand, but reduced frequencies of Treg cells on the other hand. Additionally, we demonstrate that IL-2 treatment can reverse the CREMα-mediated increases in IFN-γ producing cells and decreases in regulatory T cells in FVB/Fas–/–CREMαtg mice. An acquired IL-2 deficiency leads to disruption of the homeostasis of Treg cells and effector T cells in murine lupus models 21,28,50, while IL-2 treatment partially restores the balance in our mice. DNTs derive from CD8+ cells through down-regulation of CD8 surface co-receptors. CREMα contributes directly to the generation of DNTs by repressing CD8 transcription 37. CREMα, which is induced in response to T cell receptor (TCR) stimulation and expressed at increased levels in T cells from SLE patients, is recruited to several conserved non-coding regions within the human CD8 cluster, mediating epigenetic silencing of CD8A and CD8B 32. This suggests that the massive expansion of DNTs in FVB/Fas–/–CREMαtg mice is a direct effect of CREMα over-expression and not caused by reduced IL-2 levels in these mice. These data support the central role of CREMα in the generation of DNT cells in vivo. Consequently, IL-2 treatment did not reverse massive lymphadenopathy and splenomegaly in FVB/Fas–/–CREMαtg mice, which was clearly caused by marked expansion of DNTs. However, IL-2 administration reduced IgG antibodies significantly in sera of FVB/Fas–/–CREMαtg mice. Effects on IgG production might be caused by Treg cells themselves, as Treg cells can also directly suppress B cell antibody production in SLE 51. Treg cells thereby induce contact-dependent apoptosis in B cells in a perforin- and granzyme-dependent manner 52. It is also conceivable that enhanced levels of regulatory T cells decrease numbers of autoreactive T cells, resulting eventually in a lower production of cellular IgG in B cells.

In conclusion, our data present further evidence for a central role of CREMα in autoimmune prone conditions such as SLE. CREMα contributes to DNT generation in vivo, most probably by direct transcriptional repression of CD8. Reduced frequencies of Treg cells and enhanced frequencies of IFN-γ-producing cells can be reversed by systemic administration of IL-2. The reversibility of this homeostatic Treg cell disorder makes this a promising approach for the treatment of SLE; our data strengthen the evidence for the effectiveness of IL-2 treatment, and are in line with a recent publication from Humrich et al., who observed a rapid induction of clinical remission by low-dose IL-2 in a patient with refractory SLE 53. In particular, the data point to CREMα as a clear target for therapeutic intervention in SLE. Most interestingly, inhibition of calcium calmodulin kinase IV, which is responsible for enhanced expression of CREMα in SLE 48, improved disease pathology in a murine lupus model and in EAE 54,55. Thus, suppressing CREMα induction also provides a means of inhibiting mechanisms upstream of IL-2 instead of targeting IL-2 directly and thus represents a promising approach.

Acknowledgments

K. O. performed experiments and wrote the paper; A. W. performed experiments; A. S., N. W. and K. T. designed the study and wrote the paper. We thank Jeff Bierwagen for technical assistance. This study was supported by a grant from IZKF Aachen to K. T. (E6-7).

Disclosure

The authors declare no commercial or financial conflicts of interest.

Supporting Information

Additional Supporting Information may be found in the online version of this article.

Fig. S1. (a) Representative flow cytometry dot plots show cAMP-responsive element modulator (CREM) expression in T cells from Friend leukaemia virus, strain B/wild-type (FVB/WT) (left), FVB/Fas–/– and FVB/Fas–/–CREMαtg mice. (b) N-fold inducible cAMP early repressor (ICER) mRNA expression in splenic T cells from three FVB/WT mice compared to three FVB/CREMαtg mice (unpaired, two-tailed t-test, P = 0·021). (c) N-fold ICER mRNA expression in splenic T cells from three FVB/Fas–/– compared to three FVB/Fas–/–CREMαtg mice (unpaired, two-tailed t-test, P = 0·013).

Fig. S2. (a) Representative flow cytometry dot-plots show percentages of (a) interferon (IFN)-γ and (b) interleukin (IL)-17-positive cells in splenic CD3+CD8+ cells (black), CD3+CD4+ cells (blue) and double-negative T (DNT) cells (grey) from Friend leukaemia virus, strain B (FVB)/Fas–/– and FVB/Fas–/–cAMP-responsive element modulator (CREM)αtg mice. (c) Representative flow cytometry dot-plots show percentages of IL-2+ cells in CD3+ T cells from FVB/wild-type, FVB/CREMαtg FVB/Fas–/– and FVB/Fas–/–CREMαtg mice.

cei0181-0076-sd1.docx (704.9KB, docx)

References

  1. Crispin JC, Liossis SN, Kis-Toth K, et al. Pathogenesis of human systemic lupus erythematosus: recent advances. Trends Mol Med. 2010;16:47–57. doi: 10.1016/j.molmed.2009.12.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Tsokos GC. Systemic lupus erythematosus. N Engl J Med. 2011;365:2110–21. doi: 10.1056/NEJMra1100359. [DOI] [PubMed] [Google Scholar]
  3. Herrmann M, Voll RE, Kalden JR. Etiopathogenesis of systemic lupus erythematosus. Immunol Today. 2000;21:424–6. doi: 10.1016/s0167-5699(00)01675-3. [DOI] [PubMed] [Google Scholar]
  4. Rauen T, Hedrich CM, Tenbrock K, Tsokos GC. cAMP responsive element modulator: a critical regulator of cytokine production. Trends Mol Med. 2013;19:262–9. doi: 10.1016/j.molmed.2013.02.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Cesare D, Sassone-Corsi P. Transcriptional regulation by cyclic AMP-responsive factors. Prog Nucleic Acid Res Mol Biol. 2000;64:343–69. doi: 10.1016/s0079-6603(00)64009-6. [DOI] [PubMed] [Google Scholar]
  6. Sassone-Corsi P. Transcription factors responsive to cAMP. Annu Rev Cell Dev Biol. 1995;11:355–77. doi: 10.1146/annurev.cb.11.110195.002035. [DOI] [PubMed] [Google Scholar]
  7. Foulkes NS, Borrelli E, Sassone-Corsi P. CREM gene: use of alternative DNA-binding domains generates multiple antagonists of cAMP-induced transcription. Cell. 1991;64:739–49. doi: 10.1016/0092-8674(91)90503-q. [DOI] [PubMed] [Google Scholar]
  8. Lippe R, Ohl K, Varga G, et al. CREMalpha overexpression decreases IL-2 production, induces a T(H)17 phenotype and accelerates autoimmunity. J Mol Cell Biol. 2012;4:121–3. doi: 10.1093/jmcb/mjs004. [DOI] [PubMed] [Google Scholar]
  9. Verjans E, Ohl K, Yu Y, et al. Overexpression of CREMalpha in T cells aggravates lipopolysaccharide-induced acute lung injury. J Immunol. 2013;191:1316–23. doi: 10.4049/jimmunol.1203147. [DOI] [PubMed] [Google Scholar]
  10. Takahashi T, Tagami T, Yamazaki S, et al. Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med. 2000;192:303–10. doi: 10.1084/jem.192.2.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Thornton AM, Shevach EM. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med. 1998;188:287–96. doi: 10.1084/jem.188.2.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McNally A, Hill GR, Sparwasser T, Thomas R, Steptoe RJ. CD4+CD25+ regulatory T cells control CD8+ T-cell effector differentiation by modulating IL-2 homeostasis. Proc Natl Acad Sci USA. 2011;108:7529–34. doi: 10.1073/pnas.1103782108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lim HW, Hillsamer P, Banham AH, Kim CH. Cutting edge: direct suppression of B cells by CD4+ CD25+ regulatory T cells. J Immunol. 2005;175:4180–3. doi: 10.4049/jimmunol.175.7.4180. [DOI] [PubMed] [Google Scholar]
  14. Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol. 2003;4:337–42. doi: 10.1038/ni909. [DOI] [PubMed] [Google Scholar]
  15. Brunkow ME, Jeffery EW, Hjerrild KA, et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet. 2001;27:68–73. doi: 10.1038/83784. [DOI] [PubMed] [Google Scholar]
  16. Bennett CL, Christie J, Ramsdell F, et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet. 2001;27:20–1. doi: 10.1038/83713. [DOI] [PubMed] [Google Scholar]
  17. Wildin RS, Ramsdell F, Peake J, et al. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet. 2001;27:18–20. doi: 10.1038/83707. [DOI] [PubMed] [Google Scholar]
  18. Alexander T, Sattler A, Templin L, et al. Foxp3+ Helios+ regulatory T cells are expanded in active systemic lupus erythematosus. Ann Rheum Dis. 2013;72:1549–58. doi: 10.1136/annrheumdis-2012-202216. [DOI] [PubMed] [Google Scholar]
  19. Golding A, Hasni S, Illei G, Shevach EM. The percentage of Foxp3 Helios T regulatory cells positively correlates with disease activity in systemic lupus erythematosus. Arthritis Rheum. 2013;65:2898–906. doi: 10.1002/art.38119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Engler JB, Undeutsch R, Kloke L, et al. Unmasking of autoreactive CD4 T cells by depletion of CD25 regulatory T cells in systemic lupus erythematosus. Ann Rheum Dis. 2011;70:2176–83. doi: 10.1136/ard.2011.153619. [DOI] [PubMed] [Google Scholar]
  21. Humrich JY, Morbach H, Undeutsch R, et al. Homeostatic imbalance of regulatory and effector T cells due to IL-2 deprivation amplifies murine lupus. Proc Natl Acad Sci USA. 2010;107:204–9. doi: 10.1073/pnas.0903158107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Dolff S, Bijl M, Huitema MG, Limburg PC, Kallenberg CG, Abdulahad WH. Disturbed Th1, Th2, Th17 and T(reg) balance in patients with systemic lupus erythematosus. Clin Immunol. 2011;141:197–204. doi: 10.1016/j.clim.2011.08.005. [DOI] [PubMed] [Google Scholar]
  23. Scalapino KJ, Tang Q, Bluestone JA, Bonyhadi ML, Daikh DI. Suppression of disease in New Zealand Black/New Zealand White lupus-prone mice by adoptive transfer of ex vivo expanded regulatory T cells. J Immunol. 2006;177:1451–9. doi: 10.4049/jimmunol.177.3.1451. [DOI] [PubMed] [Google Scholar]
  24. Weigert O, von Spee C, Undeutsch R, Kloke L, Humrich JY, Riemekasten G. CD4+Foxp3+ regulatory T cells prolong drug-induced disease remission in (NZBxNZW) F1 lupus mice. Arthritis Res Ther. 2013;15:R35. doi: 10.1186/ar4188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol. 2005;6:1142–51. doi: 10.1038/ni1263. [DOI] [PubMed] [Google Scholar]
  26. Setoguchi R, Hori S, Takahashi T, Sakaguchi S. Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J Exp Med. 2005;201:723–35. doi: 10.1084/jem.20041982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. D'Cruz LM, Klein L. Development and function of agonist-induced CD25+Foxp3+ regulatory T cells in the absence of interleukin 2 signaling. Nat Immunol. 2005;6:1152–9. doi: 10.1038/ni1264. [DOI] [PubMed] [Google Scholar]
  28. Yang CH, Tian L, Ling GS, et al. Immunological mechanisms and clinical implications of regulatory T cell deficiency in a systemic autoimmune disorder: roles of IL-2 versus IL-15. Eur J Immunol. 2008;38:1664–76. doi: 10.1002/eji.200838190. [DOI] [PubMed] [Google Scholar]
  29. Hayashi T, Hasegawa K, Adachi C. Elimination of CD4(+)CD25(+) T cell accelerates the development of glomerulonephritis during the preactive phase in autoimmune-prone female NZB x NZW F mice. Int J Exp Pathol. 2005;86:289–96. doi: 10.1111/j.0959-9673.2005.00438.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wofsy D, Ledbetter JA, Hendler PL, Seaman WE. Treatment of murine lupus with monoclonal anti-T cell antibody. J Immunol. 1985;134:852–7. [PubMed] [Google Scholar]
  31. Solomou EE, Juang YT, Gourley MF, Kammer GM, Tsokos GC. Molecular basis of deficient IL-2 production in T cells from patients with systemic lupus erythematosus. J Immunol. 2001;166:4216–22. doi: 10.4049/jimmunol.166.6.4216. [DOI] [PubMed] [Google Scholar]
  32. Hedrich CM, Crispin JC, Rauen T, et al. cAMP responsive element modulator (CREM) alpha mediates chromatin remodeling of CD8 during the generation of CD3+ CD4– CD8– T cells. J Biol Chem. 2014;289:2361–70. doi: 10.1074/jbc.M113.523605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Perry D, Sang A, Yin Y, Zheng YY, Morel L. Murine models of systemic lupus erythematosus. J Biomed Biotechnol. 2011;2011:271694. doi: 10.1155/2011/271694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shirai T, Abe M, Yagita H, Okumura K, Morse HC, III, Davidson WF. The expanded populations of CD4–CD8– T cell receptor alpha/beta+ T cells associated with the lpr and gld mutations are CD2. J Immunol. 1990;144:3756–61. [PubMed] [Google Scholar]
  35. Crispin JC, Oukka M, Bayliss G, et al. Expanded double negative T cells in patients with systemic lupus erythematosus produce IL-17 and infiltrate the kidneys. J Immunol. 2008;181:8761–6. doi: 10.4049/jimmunol.181.12.8761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shivakumar S, Tsokos GC, Datta SK. T cell receptor alpha/beta expressing double-negative (CD4–/CD8–) and CD4+ T helper cells in humans augment the production of pathogenic anti-DNA autoantibodies associated with lupus nephritis. J Immunol. 1989;143:103–12. [PubMed] [Google Scholar]
  37. Hedrich CM, Rauen T, Crispin JC, et al. cAMP-responsive element modulator alpha (CREMalpha) trans-represses the transmembrane glycoprotein CD8 and contributes to the generation of CD3+CD4–CD8– T cells in health and disease. J Biol Chem. 2013;288:31880–7. doi: 10.1074/jbc.M113.508655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Balomenos D, Rumold R, Theofilopoulos AN. Interferon-gamma is required for lupus-like disease and lymphoaccumulation in MRL-lpr mice. J Clin Invest. 1998;101:364–71. doi: 10.1172/JCI750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Amarilyo G, Lourenco EV, Shi FD, La Cava A. IL-17 promotes murine lupus. J Immunol. 2014;193:540–3. doi: 10.4049/jimmunol.1400931. [DOI] [PubMed] [Google Scholar]
  40. Rosenblum MD, Gratz IK, Paw JS, Abbas AK. Treating human autoimmunity: current practice and future prospects. Sci Transl Med. 2012;4:125sr1. doi: 10.1126/scitranslmed.3003504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Altman A, Theofilopoulos AN, Weiner R, Katz DH, Dixon FJ. Analysis of T cell function in autoimmune murine strains. Defects in production and responsiveness to interleukin 2. J Exp Med. 1981;154:791–808. doi: 10.1084/jem.154.3.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wofsy D, Murphy ED, Roths JB, Dauphinee MJ, Kipper SB, Talal N. Deficient interleukin 2 activity in MRL/Mp and C57BL/6J mice bearing the lpr gene. J Exp Med. 1981;154:1671–80. doi: 10.1084/jem.154.5.1671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Linker-Israeli M, Bakke AC, Kitridou RC, Gendler S, Gillis S, Horwitz DA. Defective production of interleukin 1 and interleukin 2 in patients with systemic lupus erythematosus (SLE) J Immunol. 1983;130:2651–5. [PubMed] [Google Scholar]
  44. de Faucal P, Godard A, Peyrat MA, Moreau JF, Soulillou JP. Impaired IL2 production by lymphocytes of patients with systemic lupus erythematosus. Ann Immunol (Paris) 1984;135D:161–72. doi: 10.1016/s0769-2625(84)81108-3. [DOI] [PubMed] [Google Scholar]
  45. Alcocer-Varela J, Alarcon-Segovia D. Decreased production of and response to interleukin-2 by cultured lymphocytes from patients with systemic lupus erythematosus. J Clin Invest. 1982;69:1388–92. doi: 10.1172/JCI110579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tenbrock K, Juang YT, Tolnay M, Tsokos GC. The cyclic adenosine 5'-monophosphate response element modulator suppresses IL-2 production in stimulated T cells by a chromatin-dependent mechanism. J Immunol. 2003;170:2971–6. doi: 10.4049/jimmunol.170.6.2971. [DOI] [PubMed] [Google Scholar]
  47. Tenbrock K, Juang YT, Gourley MF, Nambiar MP, Tsokos GC. Antisense cyclic adenosine 5'-monophosphate response element modulator up-regulates IL-2 in T cells from patients with systemic lupus erythematosus. J Immunol. 2002;169:4147–52. doi: 10.4049/jimmunol.169.8.4147. [DOI] [PubMed] [Google Scholar]
  48. Juang YT, Rauen T, Wang Y, et al. Transcriptional activation of the cAMP-responsive modulator promoter in human T cells is regulated by protein phosphatase 2A-mediated dephosphorylation of SP-1 and reflects disease activity in patients with systemic lupus erythematosus. J Biol Chem. 2011;286:1795–801. doi: 10.1074/jbc.M110.166785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Juang YT, Wang Y, Solomou EE, et al. SLE serum IgG increases CREM binding to the IL-2 promoter and suppress IL-2 production through CaMKIV. FASEB J. 2005;19:A387. doi: 10.1172/JCI200522854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Mizui M, Koga T, Lieberman LA, et al. IL-2 protects lupus-prone mice from multiple end-organ damage by limiting CD4-CD8- IL-17-producing T cells. J Immunol. 2014;193:2168–77. doi: 10.4049/jimmunol.1400977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Iikuni N, Lourenco EV, Hahn BH, La Cava A. Cutting edge: regulatory T cells directly suppress B cells in systemic lupus erythematosus. J Immunol. 2009;183:1518–22. doi: 10.4049/jimmunol.0901163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zhao DM, Thornton AM, DiPaolo RJ, Shevach EM. Activated CD4+CD25+ T cells selectively kill B lymphocytes. Blood. 2006;107:3925–32. doi: 10.1182/blood-2005-11-4502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Humrich JY, von Spee-Mayer C, Siegert E, et al. Rapid induction of clinical remission by low-dose interleukin-2 in a patient with refractory SLE. Ann Rheumatic Dis. 2015;74:791–2. doi: 10.1136/annrheumdis-2014-206506. [DOI] [PubMed] [Google Scholar]
  54. Koga T, Hedrich CM, Mizui M, et al. CaMK4-dependent activation of AKT/mTOR and CREM-alpha underlies autoimmunity-associated Th17 imbalance. J Clin Invest. 2014;124:2234–45. doi: 10.1172/JCI73411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Koga T, Mizui M, Yoshida N, et al. KN-93, an inhibitor of calcium/calmodulin-dependent protein kinase IV, promotes generation and function of Foxp3 regulatory T cells in MRL/lpr mice. Autoimmunity. 2014;47:445–50. doi: 10.3109/08916934.2014.915954. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. (a) Representative flow cytometry dot plots show cAMP-responsive element modulator (CREM) expression in T cells from Friend leukaemia virus, strain B/wild-type (FVB/WT) (left), FVB/Fas–/– and FVB/Fas–/–CREMαtg mice. (b) N-fold inducible cAMP early repressor (ICER) mRNA expression in splenic T cells from three FVB/WT mice compared to three FVB/CREMαtg mice (unpaired, two-tailed t-test, P = 0·021). (c) N-fold ICER mRNA expression in splenic T cells from three FVB/Fas–/– compared to three FVB/Fas–/–CREMαtg mice (unpaired, two-tailed t-test, P = 0·013).

Fig. S2. (a) Representative flow cytometry dot-plots show percentages of (a) interferon (IFN)-γ and (b) interleukin (IL)-17-positive cells in splenic CD3+CD8+ cells (black), CD3+CD4+ cells (blue) and double-negative T (DNT) cells (grey) from Friend leukaemia virus, strain B (FVB)/Fas–/– and FVB/Fas–/–cAMP-responsive element modulator (CREM)αtg mice. (c) Representative flow cytometry dot-plots show percentages of IL-2+ cells in CD3+ T cells from FVB/wild-type, FVB/CREMαtg FVB/Fas–/– and FVB/Fas–/–CREMαtg mice.

cei0181-0076-sd1.docx (704.9KB, docx)

Articles from Clinical and Experimental Immunology are provided here courtesy of British Society for Immunology

RESOURCES