MRL/Mp CD4+,CD25− T cells show reduced sensitivity to suppression by CD4+,CD25+ regulatory T cells in vitro: A novel defect of T cell regulation in systemic lupus erythematosus

Objective

To investigate the hypothesis that loss of suppression mediated by peripheral CD4+,CD25+ regulatory T cells is a hallmark of systemic lupus erythematosus (SLE).

Methods

Mice of the MRL/Mp strain were studied as a polygenic model of SLE. Following immunomagnetic selection, peripheral lymphoid CD25+ and CD25− CD4+ T cells were cultured independently or together in the presence of anti‐CD3/CD28 monoclonal antibody–coated beads. Proliferation was assessed by measuring the incorporation of tritiated thymidine.

Results

While MRL/Mp CD4+,CD25+ regulatory T cells showed only subtle abnormalities of regulatory function in vitro, syngeneic CD4+,CD25− T cells showed significantly reduced sensitivity to suppression, as determined by crossover experiments in which MRL/Mp CD4+,CD25− T cells were cultured with H‐2–matched CBA/Ca CD4+,CD25+ regulatory T cells in the presence of a polyclonal stimulus.

Conclusion

Our findings highlight a novel defect of peripheral tolerance in SLE. Identification of this defect could open new opportunities for therapeutic intervention.

     

MicroRNA‐126 regulates DNA methylation in CD4+ T cells and contributes to systemic lupus erythematosus by targeting DNA methyltransferase 1

Objective

To identify microRNA genes with abnormal expression in the CD4+ T cells of patients with systemic lupus erythematosus (SLE) and to determine the role of microRNA‐126 (miR‐126) in the etiology of SLE.

Methods

MicroRNA expression patterns in CD4+ T cells from patients with SLE and healthy control subjects were analyzed by microRNA microarray and stem loop quantitative polymerase chain reaction (qPCR). Luciferase reporter gene assays were performed to identify miR‐126 targets. Dnmt1, CD11a, and CD70 messenger RNA and protein levels were determined by real‐time qPCR, Western blotting, and flow cytometry. CD11a, CD70, and EGFL7 promoter methylation levels were detected by bisulfite sequencing. IgG levels in T cell–B cell cocultures were determined by enzyme‐linked immunosorbent assay.

Results

The expression of 11 microRNA was significantly increased or decreased in CD4+ T cells from patients with SLE relative to that in CD4+ T cells from control subjects. Among these, miR‐126 was up‐regulated, and its degree of overexpression was inversely correlated with Dnmt1 protein levels. We demonstrated that miR‐126 directly inhibits Dnmt1 translation via interaction with its 3′–untranslated region, and that overexpression of miR‐126 in CD4+ T cells can significantly reduce Dnmt1 protein levels. The overexpression of miR‐126 in CD4+ T cells from healthy donors caused the demethylation and up‐regulation of genes encoding CD11a and CD70, thereby causing T cell and B cell hyperactivity. The inhibition of miR‐126 in CD4+ T cells from patients with SLE had the opposite effects. Expression of the miR‐126 host gene EGFL7 was also up‐regulated in CD4+ T cells from patients with SLE, possibly in a hypomethylation‐dependent manner.

Conclusion

Our data suggest that miR‐126 regulates DNA methylation in CD4+ T cells and contributes to T cell autoreactivity in SLE by directly targeting Dnmt1.

     

Lower expression levels of the programmed death 1 receptor on CD4+CD25+ T cells and correlation with the PD‐1.3A genotype in patients with systemic lupus erythematosus

Objective

A genetic polymorphism in the programmed death 1 (PD‐1) gene encoding the coinhibitory PD‐1 immunoreceptor, PD‐1.3A, is associated with systemic lupus erythematosus (SLE). The aim of this study was to assess PD‐1 receptor expression in patients with SLE, in comparison with relatives and unrelated healthy controls, and to identify correlations of lower expression levels of PD‐1 receptor with the PD‐1.3A genotype.

Methods

Patients with SLE, patients' relatives, and unrelated healthy control subjects from Iceland and Sweden were studied. Peripheral blood mononuclear cells (PBMCs) were stimulated with anti‐CD3/anti‐CD28, and PD‐1 expression was analyzed by flow cytometry. PD‐1.3A/G genotyping was performed using polymerase chain reaction–restriction fragment length polymorphism analysis.

Results

PD‐1 expression on PBMCs was induced after antibody stimulation, showing increases of 2.1‐fold in SLE patients, 3.1‐fold in relatives, and 5.1‐fold in healthy controls. The frequency of PD‐1+ cells was significantly lower in SLE patients compared with relatives and healthy controls. PD‐1 expression on PD‐1+ cells and on CD4+CD25+ T cells was significantly lower in SLE patients and relatives compared with healthy controls. PD‐1 expression was significantly elevated on CD25^high cells. Levels of PD‐1 expression on CD25^high and CD25^intermediate cells were significantly lower in SLE patients compared with healthy controls. PD‐1 was expressed on both FoxP3− and FoxP3+ cells. Lower expression of PD‐1 was significantly correlated with the PD‐1.3A/G genotype.

Conclusion

The results demonstrate significantly lower PD‐1 receptor expression in SLE patients and their relatives and reveal a significant correlation of lower PD‐1 expression with the PD‐1.3A allele. Thus, PD‐1.3A may contribute to abnormalities in PD‐1 receptor expression on CD4+CD25+ T cells in patients with SLE, providing support for an important role of the PD‐1 pathway in SLE and, possibly, in other autoimmune diseases.

     

Differential control of CD22 ligand expression on B and T lymphocytes,and enhanced expression in murine systemic lupus

Objective

CD22, a B cell–restricted transmembrane glycoprotein, regulates B cell antigen receptor signaling upon interaction with α2,6‐linked sialic acid–bearing glycans, which act as ligands and are expressed on B and T cells. In this study, we investigated how the expression of CD22 ligand (CD22L) is modulated following lymphocyte activation or during the course of systemic lupus erythematosus (SLE).

Methods

The expression levels of CD22L on B and T cells in nonautoimmune mice were assessed by flow cytometric analysis using a soluble recombinant form of CD22, following stimulation with antigen or mitogen in vitro. In addition, the expression levels of CD22L on circulating lymphocytes were correlated with the progression of SLE in lupus‐prone mice.

Results

We observed a constitutive expression of CD22L on mature B cells, but not T cells, in nonautoimmune mice. However, CD22L levels were up‐regulated selectively on T cells (but not B cells) stimulated with antigens in vitro, while their expression levels on B cells was up‐modulated following polyclonal activation with lipopolysaccharide. Furthermore, expression of CD22L was increased on circulating B cells (and to a lesser extent on T cells) in parallel with progression of SLE in several different lupus‐prone mice and in a cohort of (C57BL/6 × [NZB × C57BL/6.Yaa]F₁) backcross mice.

Conclusion

The expression of CD22L is differentially regulated in B and T cells, and high expression of CD22L on circulating B cells is a marker for development of severe SLE, suggesting a role for CD22–CD22L interactions in SLE as well as in the regulation of humoral immunity.

     

Dysregulated expression of CXCR4/CXCL12 in subsets of patients with systemic lupus erythematosus

Objective

CXCR4 is a chemokine with multiple effects on the immune system. In murine lupus models, we demonstrated that monocytes, neutrophils, and B cells overexpressed CXCR4 and that its ligand, CXCL12, was up‐regulated in diseased kidneys. We undertook this study to determine whether CXCR4 expression was increased in peripheral blood leukocytes from patients with systemic lupus erythematosus (SLE) and whether CXCL12 expression was increased in kidneys from patients with SLE.

Methods

Peripheral blood leukocytes from 31 SLE patients, 8 normal controls, and 9 patients with rheumatoid arthritis were prospectively analyzed by flow cytometry for CXCR4 expression. Biopsy samples (n = 14) from patients with lupus nephritis (LN) were immunostained with anti‐CXCL12 antibody.

Results

CD19+ B cells and CD4+ T cells from SLE patients displayed a >2‐fold increase (P = 0.0001) and >3‐fold increase (P < 0.0001), respectively, in median CXCR4 expression compared with that in controls (n = 7–8). Moreover, CXCR4 expression on B cells was 1.61‐fold higher in patients with SLE Disease Activity Index (SLEDAI) scores >10 (n = 8) than in patients with SLEDAI scores ≤10 (n = 16) (P = 0.0008), 1.71‐fold higher in patients with class IV LN (n = 5) than in patients with other classes of LN (n = 7) (P = 0.02), and 1.40‐fold higher in patients with active neuropsychiatric SLE (NPSLE) (n = 6) than in patients with inactive NPSLE (n = 18) (P = 0.01). CXCL12 was significantly up‐regulated in the tubules and glomeruli of kidneys in patients with LN (n = 14), with the percentage of positive cells correlating positively with the severity of LN.

Conclusion

CXCR4 appears to be up‐regulated in multiple leukocyte subsets in SLE patients. The heightened expression of CXCR4 on B cells in active NPSLE and of CXCL12 in nephritic kidneys suggests that the CXCR4/CXCL12 axis might be a potential therapeutic target for SLE patients with kidney and/or central nervous system involvement.

     

Decreased microRNA‐142‐3p/5p expression causes CD4+ T cell activation and B cell hyperstimulation in systemic lupus erythematosus

Objective

To examine the role of microRNA‐142‐3p/5p (miR‐142‐3p/5p) in the development of autoimmunity in patients with systemic lupus erythematosus (SLE).

Methods

MicroRNA‐142‐3p/5p expression levels were determined by real‐time quantitative polymerase chain reaction, and potential target genes were verified using luciferase reporter gene assays. The effects of miR‐142‐3p/5p on T cell function were assessed by transfection with miR‐142‐3p/5p inhibitors or mimics. Histone modifications and methylation levels within a putative regulatory region of the miR‐142 locus were detected by chromatin immunoprecipitation assay and bisulfite sequencing, respectively.

Results

We confirmed that miR‐142‐3p and miR‐142‐5p were significantly down‐regulated in SLE CD4+ T cells compared with healthy controls and observed that miR‐142‐3p/5p levels were inversely correlated with the putative SLE‐related targets signaling lymphocytic activation molecule–associated protein (SAP), CD84, and interleukin‐10 (IL‐10). We demonstrated that miR‐142‐3p and miR‐142‐5p directly inhibit SAP, CD84, and IL‐10 translation, and that reduced miR‐142‐3p/5p expression in CD4+ T cells can significantly increase protein levels of these target genes. Furthermore, inhibiting miR‐142‐3p/5p in healthy donor CD4+ T cells caused T cell overactivation and B cell hyperstimulation, whereas overexpression of miR‐142‐3p/5p in SLE CD4+ T cells had the opposite effect. We also observed that the decrease in miR‐142 expression in SLE CD4+ T cells correlated with changes to histone modifications and DNA methylation levels upstream of the miR‐142 precursor sequence.

Conclusion

The results of this study indicate that reduced expression of miR‐142‐3p/5p in the CD4+ T cells of patients with SLE causes T cell activity and B cell hyperstimulation.

     

The response of T cells to interleukin‐6 is differentially regulated by the microenvironment of the rheumatoid synovial fluid and tissue

Objective

Interleukin‐6 (IL‐6) is a proinflammatory cytokine with regulatory effects on the survival and differentiation of T cells. It exerts its biologic function in 2 ways: by directly binding to the IL‐6 receptor (IL‐6R; CD126) or via trans‐signaling, in which soluble IL‐6R/IL‐6 complexes bind to the signaling component CD130. This study was undertaken to assess the expression and regulation of CD126 and CD130 and determine how these affect the response of CD4+ T cells to IL‐6 in the joints of patients with rheumatoid arthritis (RA).

Methods

Flow cytometry and immunofluorescence microscopy were used to determine the expression, function, and regulation of CD126 and CD130 in CD4+ T cells from the peripheral blood (PB), synovial fluid (SF), and synovial tissue of RA patients.

Results

Compared to the findings in RA PB, CD4+ T cells in the SF and synovial tissue expressed low levels of CD126. In contrast, whereas CD4+ T cell expression of CD130 was minimal in the SF, its level in the synovial tissue was high. Consistent with this phenotype, synovial tissue T cells responded to trans‐signaling by soluble IL‐6R/IL‐6 complexes, whereas no response was evident in CD4+ T cells from the SF. Down‐regulation of both receptor components in SF T cells could be explained by exposure to high levels of IL‐6. Increased levels of CD130 messenger RNA and protein in synovial tissue CD4+ T cells suggested that CD130 is up‐regulated locally. Among a range of cytokines tested, only IL‐10 induced CD130 expression in T cells.

Conclusion

The inflamed microenvironment in the synovial tissue maintains responsiveness to IL‐6 trans‐signaling through the up‐regulation of CD130 expression in CD4+ T cells, and this process may be driven by IL‐10.

     

Interaction with activated monocytes enhances cytokine expression and suppressive activity of human CD4+CD45ro+CD25+CD127low regulatory T cells

Objective

Despite the high frequency of CD4+ T cells with a regulatory phenotype (CD25+CD127^low FoxP3+) in the joints of patients with rheumatoid arthritis (RA), inflammation persists. One possible explanation is that human Treg cells are converted into proinflammatory interleukin‐17 (IL‐17)–producing cells by inflammatory mediators and thereby lose their suppressive function. The aim of this study was to investigate whether activated monocytes, which are potent producers of inflammatory cytokines and are abundantly present in the rheumatic joint, induce proinflammatory cytokine expression in human Treg cells and impair their regulatory function.

Methods

The presence and phenotype of CD4+CD45RO+CD25+CD127^low T cells (memory Treg cells) and CD14+ monocytes in the peripheral blood (PB) and synovial fluid (SF) of patients with RA were investigated by flow cytometry. Memory Treg cells obtained from healthy control subjects underwent fluorescence‐activated cell sorting and then were cocultured with autologous activated monocytes and stimulated with anti‐CD3 monoclonal antibodies. Intracellular cytokine expression, phenotype, and function of cells were determined by flow cytometry, enzyme‐linked immunosorbent assay, and proliferation assays.

Results

In patients with RA, the frequencies of CD4+CD45RO+CD25+CD127^low Treg cells and activated CD14+ monocytes were higher in SF compared with PB. In vitro–activated monocytes induced an increase in the percentage of IL‐17–positive, interferon‐γ (IFNγ)–positive, and tumor necrosis factor α (TNFα)–positive Treg cells as well as IL‐10–positive Treg cells. The observed increase in IL‐17–positive and IFNγ‐positive Treg cells was driven by monocyte‐derived IL‐1β, IL‐6, and TNFα and was mediated by both CD14+CD16− and CD14+CD16+ monocyte subsets. Despite enhanced cytokine expression, cells maintained their CD25+FoxP3+CD39+ Treg cell phenotype and showed an enhanced capacity to suppress T cell proliferation and IL‐17 production.

Conclusion

Treg cells exposed to a proinflammatory environment show increased cytokine expression as well as enhanced suppressive activity.

     

Roles of CCR2 and CXCR3 in the T cell–mediated response occurring during lupus flares

Objective

Infiltrating lymphocytes have been demonstrated to play an important role in the tissue injury that occurs in systemic lupus erythematosus (SLE). Inflammatory chemokines control lymphocyte traffic through their interaction with T cell chemokine receptors. In this study we assessed the expression of chemokine receptors on T cell subsets of patients with active or inactive SLE.

Methods

Forty‐four SLE patients (40 women and 4 men) were included in the study. The patients were divided according to their SLE Disease Activity Index (SLEDAI), which resulted in a group of patients with inactive SLE (n = 27) and a group with active SLE (n = 17). The control group was composed of 22 healthy blood donors. A disease control group consisted of 18 patients infected with human immunodeficiency virus. Expression of chemokine receptors CCR1, CCR2, CCR5, CXCR3, CXCR4, and CX3CR1 was assessed on whole blood samples by immunofluorescence analysis.

Results

On T lymphocytes, significant differences between the SLE patients and controls were observed only in the expression of CCR2 and CXCR3. On monocytes, no significant differences in CCR2 expression were observed between the healthy controls and the SLE patients. The proportion of CD8+,CCR2+ T cells was significantly lower in the SLE patients compared with the controls (mean ± SD 2.3 ± 1.3% and 3.5 ± 3.2% in the active and inactive SLE groups, respectively, versus 21 ± 24% in controls; P < 0.0001 for both). The CD4+,CCR2+ subset was represented similarly among the controls and patients with inactive SLE (16.7 ± 5.8% and 12.8 ± 8.1%, respectively) but was depleted in patients with active SLE (7.1 ± 4.4%; P < 0.0001 versus controls). The active SLE group expressed significantly lower circulating levels of CD4+,CCR2+ T cells than did the inactive disease group (P = 0.007). A negative correlation was found between the proportion of CD4+,CCR2+ T cells and the SLEDAI (r = −0.43, P = 0.005, by Spearman's correlation). Proportions of CD8+,CXCR3+ T cells were similar between the SLE groups and the control group (58 ± 22.6% in active SLE, 47.1 ± 20% in inactive SLE, and 59.4 ± 17.3% in controls). The proportion of CXCR3‐expressing CD4+ T cells was decreased in the active disease group (23.5 ± 3.2% versus 39.9 ± 12.5% in controls; P = 0.008) but not in the inactive disease group (34.8 ± 9.5%). A trend toward a significant negative correlation was observed between the decreased proportion of CD4+,CXCR3+ T cells and the SLEDAI (P = 0.08). Following in vitro activation of purified CD4 T cells, only CCR2 was internalized, whereas expression of CXCR3 was retained in activated CD4 cells.

Conclusion

The numbers of circulating CD4+,CXCR3+ and CD4+,CCR2+ T cells are selectively decreased during SLE flares. A decrease in the number of circulating CD4+ T cells expressing CCR2 and/or CXCR3 could serve as a biomarker of the SLE flare.

     

CXCR3+CD4+ T cells are enriched in inflamed kidneys and urine and provide a new biomarker for acute nephritis flares in systemic lupus erythematosus patients

Objective

The high frequency of CD4+ T cells in interstitial infiltrates of patients with lupus nephritis suggests a contribution of these cells to local pathogenesis. The aim of this study was to examine the role of CXCR3 and the chemokine CXCL10 in recruiting these cells into the kidney and to determine whether the infiltrating T cells could be monitored in the urine to provide a reliable biomarker for acute lupus nephritis.

Methods

The frequencies of CD3+ T cells, CXCR3+ cells, and CXCL10+ cells were determined by immunohistochemical and immunofluorescence analyses of kidney sections from 18 patients with lupus nephritis. The frequency of CXCR3+CD4+ T cells was determined by flow cytometry of peripheral blood and urine from 38 patients with systemic lupus erythematosus (SLE), and the values were compared with disease activity as determined by the Systemic Lupus Erythematosus Disease Activity Index.

Results

In renal biopsy tissues from patients with lupus nephritis, a mean of 63% of the infiltrating cells expressed CXCR3, ∼60% of them were T cells, and the CXCR3+ cells colocalized with CXCL10‐producing cells. In biopsy tissues from SLE patients with acute nephritis, ∼50% of the urinary CD4+ T cells were CXCR3+, as compared with 22% in the peripheral blood, and the frequency of urinary CXCR3+CD4+ T cells correlated with disease activity. Moreover, the number of urinary CD4+ T cells reflected nephritis activity, and elevation above 800 CD4+ T cells per 100 ml of urine sharply delineated active from inactive nephritis.

Conclusion

CXCR3+ T cells are recruited into the inflamed kidneys, are enriched in the urine, and are a valuable marker of nephritis activity in SLE. They also present a potential target for future therapies.

     

Reconstitution of deficient T cell receptor ζ chain restores T cell signaling and augments T cell receptor/CD3–induced interleukin‐2 production in patients with systemic lupus erythematosus

Objective

T cells from a majority of patients with systemic lupus erythematosus (SLE) display antigen receptor–mediated signaling aberrations associated with defective T cell receptor (TCR) ζ chain, a subunit of the TCR/CD3 complex. This study was undertaken to explore the possibility that forced expression of TCR ζ chain may reverse the known signaling abnormalities and defective interleukin‐2 (IL‐2) production in SLE T cells.

Methods

Freshly isolated SLE T cells were transfected with TCR ζ chain construct in a eukaryotic expression vector at high efficiency, by a recently developed nucleoporation technique. Restoration of TCR/CD3‐mediated signaling was studied in the ζ chain–transfected cells.

Results

In SLE T cells transfected with TCR ζ chain, surface expression of TCR chain was increased and the TCR/CD3‐induced increased free intracytoplasmic calcium concentration response was normalized, as was hyperphosphorylation of cellular substrates. Simultaneously, the previously noted increased expression of the Fc receptor γ chain was diminished in SLE T cells transfected with the ζ chain expression vector, and the surface membrane clusters of cell signaling molecules were redistributed to a more continuous pattern. TCR ζ chain replacement also augmented the expression of diminished TCR/CD3‐mediated IL‐2 production in SLE T cells, associated with increased expression of the p65 subunit of nuclear factor κB in the nuclear fractions of these T cells.

Conclusion

These results suggest that reconstitution of deficient TCR ζ chain can reverse the TCR/CD3‐mediated signaling abnormalities as well as the defective IL‐2 production in T cells of patients with SLE.

     

Increased expression of CD40 on bone marrow CD34+ hematopoietic progenitor cells in patients with systemic lupus erythematosus: Contribution to Fas‐mediated apoptosis

Objective

Patients with systemic lupus erythematosus (SLE) display increased apoptosis of bone marrow (BM) CD34+ hematopoietic progenitor cells. This study was undertaken to evaluate the expression of CD40 and CD40L in the BM of SLE patients, and to explore the possible involvement of these molecules in apoptosis of CD34+ cells.

Methods

The proportion and survival characteristics of CD40+ cells within the BM CD34+ fraction from SLE patients and healthy controls were evaluated by flow cytometry. The production of CD40L by BM stromal cells was assessed using long‐term BM cultures, and the effect of CD40L on the survival characteristics and clonogenic potential of CD34+ cells was evaluated ex vivo by flow cytometry and clonogenic assays.

Results

SLE patients displayed an increased proportion of CD40+ cells within the CD34+ fraction as compared with controls. The CD34+CD40+ subpopulation contained an increased proportion of apoptotic cells compared with the CD34+CD40− fraction in patients and controls, suggesting that CD40 is involved in the apoptosis of CD34+ cells. Stimulation of patients' CD34+ cells with CD40L increased the proportion of apoptotic cells and decreased the proportion of colony‐forming cells as compared with untreated cultures. The CD40L‐mediated effects were amplified following treatment with recombinant Fas ligand, suggesting that the effects of these ligands are synergistic. CD40L levels were significantly increased in long‐term BM culture supernatants and adherent layers of BM cells from SLE patients as compared with controls.

Conclusion

These data reveal a novel role for the CD40/CD40L dyad in SLE by demonstrating that up‐regulation and induction of CD40 on BM CD34+ cells from patients with SLE contribute to the amplification of Fas‐mediated apoptosis of progenitor cells.

     

Presence of CD4+CD8+ double‐positive T cells with very high interleukin‐4 production potential in lesional skin of patients with systemic sclerosis

Objective

Fibrotic skin changes in systemic sclerosis (SSc) are preceded by the appearance of an inflammatory infiltrate rich in T cells. Since no direct comparison with T cells in normal skin has been performed previously, this study was undertaken to functionally characterize T cells in the skin of patients with early active SSc and in normal skin.

Methods

We characterized coreceptor expression, T cell receptor (TCR) usage, cytokine production, and helper and cytolytic activity of T cell lines and clones established from skin biopsy specimens from 6 SSc patients and 4 healthy individuals. Immunofluorescence analysis of skin biopsy and peripheral blood samples was performed to confirm the presence of specific subsets in vivo.

Results

A distinct subset expressing both CD4 and CD8α/β coreceptors at high levels (double‐positive [DP]) was present in T cell lines from SSc and normal skin. DP T cells actively transcribed both accessory molecules, exerted clonally distributed cytolytic and helper activity, and expressed TCR clonotypes distinct from those in CD4+ or CD8+ single‐positive (SP) T cells. In SSc skin, DP T cells produced very high levels of interleukin‐4 (IL‐4) compared with CD4+ SP T cells. Furthermore, DP T cells were directly identified in SSc skin, thus providing evidence that they are a distinct subset in vivo.

Conclusion

The present findings show that T cells with the unusual CD4+CD8+ DP phenotype are present in the skin. Their very high level of IL‐4 production in early active SSc may contribute to enhanced extracellular matrix deposition by fibroblasts.