Vasoactive intestinal peptide induces CD4+,CD25+ T regulatory cells with therapeutic effect in collagen‐induced arthritis

Objective

CD4+,CD25+ T regulatory cells (Treg) control the immune response to a variety of antigens, including self antigens, and may offer opportunities to intervene in the course of autoimmune diseases. Several models support the idea of the peripheral generation of CD4+,CD25+ Treg from CD4+,CD25− T cells, but little is known about the endogenous factors and mechanisms controlling the peripheral expansion of CD4+,CD25+ Treg. We undertook this study to investigate the capacity of the vasoactive intestinal peptide (VIP), an immunosuppressive antiarthritic neuropeptide, to induce functional Treg in vivo during the development of collagen‐induced arthritis (CIA).

Methods

We measured the number of CD4+,CD25+ Treg following VIP administration to CIA mice, and we characterized their phenotype and their ability to suppress activation of autoreactive T cells. We determined the capacity of VIP to induce Treg in vitro as well as the use of Treg in the treatment of CIA, measuring the clinical evolution and the inflammatory and autoimmune components of the disease.

Results

The administration of VIP to arthritic mice resulted in the expansion of CD4+,CD25+,Foxp3+ Treg in the periphery and joints, which inhibited autoreactive T cell activation/expansion. VIP induced more efficient suppressors on a per‐cell basis. The VIP‐generated CD4+,CD25+ Treg transfer suppressed and significantly ameliorated the progression of the disease.

Conclusion

These results demonstrate the involvement of the generation of Treg in the therapeutic effect of VIP on CIA. The generation of highly efficient Treg by VIP ex vivo could be used as an attractive therapeutic tool in the future, avoiding the administration of the peptide to patients with rheumatoid arthritis.

     

CD4+CD25+ regulatory T cells in rheumatoid arthritis: Differences in the presence,phenotype, and function between peripheral blood and synovial fluid

Objective

In mice, CD4+CD25+ regulatory T cells play a pivotal role in preventing autoimmunity. Regulatory T cells are also present and functional in healthy humans. We investigated the presence, phenotype, and function of CD4+CD25+ regulatory T cells in peripheral blood (PB) and synovial fluid (SF) from patients with rheumatoid arthritis (RA).

Methods

The presence and phenotype of CD4+CD25+ regulatory T cells were determined by flow cytometry. Anergy and suppressive activity were assessed by culturing CD4+CD25− and CD4+CD25+ T cells with anti‐CD3 monoclonal antibodies and antigen‐presenting cells, followed by proliferation and cytokine detection.

Results

The percentage of CD4+CD25+ T cells in RA SF was significantly increased compared with that in RA PB, and both of these percentages were higher than that in PB from controls. The cells in RA PB were similar in phenotype and function to CD4+CD25+ regulatory T cells from controls. In SF, however, ∼40–50% of CD4+CD25+ T cells expressed an activated phenotype, i.e., CD69+, class II MHC+, OX‐40+, with high levels of CTLA‐4 and glucocorticoid‐induced tumor necrosis factor receptor. These synovial CD4+CD25+ T cells displayed an increased suppressive capacity compared with blood CD4+CD25+ T cells. However, this enhanced suppressive activity was counterbalanced, because activated responder T cells from SF were less susceptible to CD4+CD25+ T cell–mediated suppression than were responder cells from PB.

Conclusion

We demonstrate that CD4+CD25+ regulatory T cells are present and functional in patients with RA, with higher numbers of regulatory T cells with increased suppressive activity found in SF compared with PB. These findings suggest a negative feedback system that is active at the site of inflammation. The balance between activated responder and regulatory T cells appears to influence the extent of immunoregulation in RA.

     

Proinflammatory mediator–induced reversal of CD4+,CD25+ regulatory T cell–mediated suppression in rheumatoid arthritis

Objective

We previously demonstrated that CD4+,CD25+ regulatory T (Treg) cells are present in increased numbers in the synovial fluid (SF) of rheumatoid arthritis (RA) patients and display enhanced suppressive activity as compared with their peripheral blood (PB) counterparts. Despite the presence of these immunoregulatory cells in RA, chronic inflammation persists. The purpose of the present study was to investigate whether particular proinflammatory mediators that are associated with RA could abrogate CD4+,CD25+ Treg–mediated suppression.

Methods

Monocyte phenotype was determined by flow cytometry and cytokine levels by enzyme‐linked immunosorbent assay. Magnetically sorted CD4+,CD25– and CD4+,CD25+ T cells derived from the PB and SF obtained from RA patients were stimulated alone or in coculture with anti‐CD3 monoclonal antibody (mAb) and autologous antigen‐presenting cells, in the absence or presence of anti‐CD28 mAb or the proinflammatory cytokines interleukin‐6 (IL‐6), tumor necrosis factor α (TNFα), or IL‐7.

Results

Monocytes from the SF of RA patients displayed increased expression of HLA class II molecules, CD80, CD86, and CD40 as compared with PB‐derived monocytes, indicating their activated status. Mimicking this increased costimulatory potential, addition of anti‐CD28 mAb to cocultures of CD4+,CD25– and CD4+,CD25+ T cells resulted in reduced CD4+,CD25+ Treg–mediated suppression in both PB and SF. Furthermore, IL‐7 and, to a limited extent, TNFα, both of which are produced by activated monocytes and were detected in SF, abrogated the CD4+,CD25+ Treg–mediated suppression. In contrast, IL‐6 did not influence Treg‐mediated suppression.

Conclusion

Our findings suggest that the interaction of CD4+,CD25+ Treg cells with activated monocytes in the joint might lead to diminished suppressive activity of CD4+,CD25+ Treg cells in vivo, thus contributing to the chronic inflammation in RA.

     

Effective treatment of collagen‐induced arthritis by adoptive transfer of CD25+ regulatory T cells

Objective

Regulatory T cells play an important role in the prevention of autoimmunity and have been shown to be effective in the treatment of experimental colitis, a T cell–mediated and organ‐specific disease. We previously demonstrated that intrinsic CD25+ regulatory T cells modulate the severity of collagen‐induced arthritis (CIA), which, in contrast to colitis, is a systemic antibody‐mediated disease and an accepted model of rheumatoid arthritis. We undertook this study to determine whether regulatory T cells have the potential to be used therapeutically in arthritis.

Methods

We transferred CD4+,CD25+ T cells into mice exhibiting arthritis symptoms, both immunocompetent mice and mice subjected to lethal irradiation and rescued with syngeneic bone marrow transplantation.

Results

A single transfer of regulatory T cells markedly slowed disease progression, which could not be attributed to losses of systemic type II collagen–specific T and B cell responses, since these remained unchanged after adoptive transfer. However, regulatory T cells could be found in the inflamed synovium soon after transfer, indicating that regulation may occur locally in the joint.

Conclusion

Our data indicate that CD25+ regulatory T cells can be used for the treatment of systemic, antibody‐mediated autoimmune diseases, such as CIA.

     

CD25+ cell depletion hastens the onset of severe disease in collagen‐induced arthritis

Objective

CD4+,CD25+ T regulatory cells may offer opportunities to intervene in the course of autoimmune disease. We wished to evaluate their potential for influencing systemic and chronic joint inflammation by investigating their involvement in collagen‐induced arthritis (CIA).

Methods

We depleted DBA/1 mice of CD25+ regulatory cells by injection of a depleting monoclonal antibody specific for CD25 14 days before a single immunization with type II collagen (CII) in Freund's complete adjuvant. CD4+,CD25+ T cells were adoptively transferred to some groups of mice during immunization. Mice were then scored for signs of arthritis, and blood was taken periodically to measure the amounts of CII‐specific antibodies. Splenocytes of treated mice were examined in vitro to determine the effects of depletion on proliferation to CII and control antigens.

Results

CD25+ cell–depleted DBA/1 mice had significantly more severe disease than control mice following collagen immunization. The magnified severity was also accompanied by higher antibody titers against collagen, and in vitro tests showed increased proliferation of collagen‐specific T cells. Adoptively transferring CD4+,CD25+ T cells into depleted mice was shown to reverse the heightened severity. Control mice, which were depleted and immunized with the neoantigen keyhole limpet hemocyanin (KLH), had neither an increased antibody response toward KLH nor an augmented proliferative response, indicating that CD25+ cell depletion preferentially affects immunity against self antigen.

Conclusion

These results establish a link between CD4+,CD25+ regulatory cells and CIA and provide a rationale for investigating CD4+,CD25+ T regulatory cells in the treatment and prevention of arthritis.

     

CD4+,CD28− T cells in rheumatoid arthritis patients combine features of the innate and adaptive immune systems

Objective

To determine whether CD4+,CD28− T cells, which are expanded in patients with rheumatoid arthritis (RA), express receptors that typically regulate the function of natural killer (NK) cells.

Methods

Expression of the NK cell surface molecules CD158, p70, CD94, CD161, and CD8α on T cell subsets was determined by multicolor flow cytometric analysis of peripheral blood mononuclear cells from 36 RA patients. Expression of CD161 on tissue‐infiltrating CD4 T cells was determined by 2‐color immunohistochemistry analysis of synovial tissue samples.

Results

Killer cell–inhibitory receptors (KIR) and killer cell–activating receptors (KAR) were exclusively expressed on CD4+,CD28− T cells, with the CD158b molecule being the most frequently detected isoform. A coordinated mechanism inducing KIR/KAR expression was suggested by similarities in the expression of CD158b on CD4 and CD8 T cells. CD4+,CD28− T cells were also positive for CD8‐αα homodimers, another characteristic shared with NK cells. Of the C‐type lectin NK cell receptors (NK receptors), CD94 was consistently absent, but CD161 was found on a CD4 T cell population that is significantly expanded in RA patients (P = 0.01). Involvement in disease of NK receptor–expressing CD4 T cells was suggested by the presence of CD4+,CD161+ T cells in follicular microstructures typical of rheumatoid synovitis.

Conclusion

Patients with RA have an expanded and unusual subset of CD4 T cells that infiltrates the tissue lesions and is characterized by a deficiency of CD28, the expression of CD8‐αα homodimers, and the expression of several types of HLA class I–recognizing NK receptors. CD4 T cells bearing NK receptors can bridge functions of the innate and adaptive immune systems, such as responsiveness to specific antigen, rapid release of interferon‐γ, cytotoxicity, independence from classic costimulatory pathways, and integration of multiple activating and inhibitory signals to control effector functions.

     

Impaired suppression of synovial fluid CD4+CD25− T cells from patients with juvenile idiopathic arthritis by CD4+CD25+ Treg cells

Objective

Natural CD4+CD25+FoxP3+ Treg cells play a crucial role in maintaining immune homeostasis and controlling autoimmunity. In patients with juvenile idiopathic arthritis (JIA), inflammation occurs despite the increased total numbers of Treg cells in the synovial fluid (SF) compared to the peripheral blood (PB). This study was undertaken to investigate the phenotype of CD4+ T cells in PB and SF from JIA patients, the function of synovial Treg cells, and the sensitivity of PB and SF CD4+CD25− effector T cells to the immunoregulatory properties of Treg cells, and to study the suppression of cytokine secretion from SF effector T cells by Treg cells.

Methods

The phenotypes of effector T cells and Treg cells of PB and SF from JIA patients and healthy donors were determined by flow cytometry. The functionality of isolated Treg cells and effector T cells was quantified in ³H‐thymidine proliferation assays. Cytokine levels were analyzed using Bio‐Plex Pro assay.

Results

Compared to PB, SF showed significantly elevated numbers of activated and differentiated CD4+CD45RO+ T cells. Sensitivity of SF effector T cells to the suppressive effects of Treg cells from both PB and SF was impaired, correlating inversely with the expression of CD69 and HLA–DR. However, SF effector T cell cytokine secretion was partly suppressed by SF Treg cells.

Conclusion

Our findings indicate that regulation is impaired in the SF of patients with JIA, as shown by the resistance of effector T cells to immunoregulation by functional Treg cells. This resistance of the SF effector T cells might be due to their activated phenotype.

     

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.

     

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.

     

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.     

Expression of CD44 variant isoforms CD44v3 and CD44v6 is increased on T cells from patients with systemic lupus erythematosus and is correlated with disease activity

Objective

To quantify the expression of CD44 and variant isoforms CD44v3 and CD44v6 on T cells from patients with systemic lupus erythematosus (SLE), and to assess correlations of the level of expression of these molecules with disease manifestations.

Methods

Information on clinical and demographic characteristics was collected, and blood samples were obtained from 72 patients with SLE and 32 healthy control subjects matched to the patients by sex, race, and age. Expression of CD44 and variants CD44v3 and v6 on T cell subsets was determined by flow cytometry, and Pearson's correlations of their expression levels with clinical variables, SLE Disease Activity Index (SLEDAI) scores, and presence of lupus nephritis were determined. Wilcoxon's rank sum tests and conditional multivariable regression analyses were applied to identify differences in the expression of CD44 between patients with SLE and healthy controls.

Results

Expression of CD44 was higher on CD4+ and CD8+ T cells from SLE patients compared with controls (P ≤ 0.03). Expression of CD44v3 and CD44v6 was also higher on total T cells and CD4+ and CD8+ T cells from SLE patients compared with controls (P ≤ 0.03). Cell surface levels of CD44v3 on total T cells, CD4+ T cells, and CD8+ T cells as well as cell surface expression of CD44v6 on total T cells and CD4+ T cells were correlated with the SLEDAI score (P < 0.05). The presence of lupus nephritis was associated with the expression of CD44v6 on total T cells, CD4+ T cells, and CD4−CD8− T cells (P < 0.05). Positivity for anti–double‐stranded DNA antibodies was associated with the expression levels of CD44v6 on T cells (P < 0.05).

Conclusion

These results indicate that expression levels of CD44v3 and CD44v6 on T cells may represent useful biomarkers of SLE activity.

     

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.

     

Macrophage subpopulations in rheumatoid synovium: Reduced CD163 expression in CD4+ T lymphocyte–rich microenvironments

Objective

The cell surface glycoprotein CD163 is a member of the cysteine‐rich scavenger receptor family, highly specific for leukocytes of the mononuclear phagocyte lineage. In vitro, it is induced by glucocorticoids, interleukin‐6 (IL‐6), and IL‐10 and down‐regulated by interferon‐γ (IFNγ), indicating that it has a role in antiinflammatory or other immunomodulatory pathways. We assessed CD163 expression in microenvironments within rheumatoid arthritis (RA) synovium to clarify the relationships among CD4+ T lymphocytes, IFNγ, and macrophage function in RA.

Methods

Double immunofluorescence and serial immunoenzymatic studies were performed on normal, osteoarthritic, and RA synovium and tonsil with antibodies to CD163, CD45, CD68, CD14, CD3, CD4, CD8, CD19, and IFNγ.

Results

CD163 was observed on all CD14+ cells in synovium and tonsil with the exception of cells within larger T lymphocyte clusters in synovium and within tonsillar follicles. All brightly CD14+ cells in or around vessel walls (interpreted as immigrant monocytes) were CD163+. CD163 labeled fewer cells than did CD68 in synovial intima, but all CD45+ intimal cells were CD163+. CD4+,IFNγ+ T lymphocytes in RA synovium were chiefly localized within clusters containing CD68+, CD163− cells.

Conclusion

Within RA synovium, CD163 has major advantages as a macrophage marker and does not appear to be restricted to “mature” macrophages. CD163 discriminates between synovial macrophages and synovial intimal fibroblasts, which also stain positively for CD68 in diseased tissue.

     

Antigen‐induced,tolerogenic CD11c+,CD11b+ dendritic cells are abundant in Peyer's patches during the induction of oral tolerance to type II collagen and suppress experimental collagen‐induced arthritis

Objective

Although oral tolerance is a well‐known phenomenon, the role of dendritic cells (DCs) is not well characterized. This study was conducted to better understand the differential role played by each Peyer's patch DC subset in the induction of oral tolerance to type II collagen (CII) in murine collagen‐induced arthritis (CIA).

Methods

CII was fed 6 times to DBA/1 mice beginning 2 weeks before immunization, and the effect on arthritis was assessed. We compared the proportion of CD11c+,CD11b+ DCs and CD11c+,CD8α+ DCs in the Peyer's patches of CII‐fed tolerized and phosphate buffered saline–fed nontolerized mice after the induction of CIA. The immunosuppressive properties of each DC subset were determined using fluorescence‐activated cell sorter analysis for intracellular interleukin‐10 (IL‐10) and IL‐12 and mixed lymphocyte culture. The ability of each DC subset to induce CD4+,CD25+ T regulatory cells was also examined. Mice were injected with CII‐pulsed CD11c+,CD11b+ DCs isolated from Peyer's patches of tolerized mice, and the effect on CIA was examined.

Results

The severity of arthritis was significantly lower in tolerized mice. The proportion of CD11c+,CD11b+ DCs was increased in the Peyer's patches of tolerized mice and those DCs exhibited immunosuppressive characteristics, such as increased IL‐10 production, inhibition of T cell proliferative responses to CII, and CD4+,CD25+ regulatory T cell induction. Furthermore, the CD11c+,CD11b+ DCs suppressed the severity of arthritis upon adoptive transfer.

Conclusion

Our observations demonstrate that CD11c+,CD11b+ DCs, which are abundant in Peyer's patches during the induction of oral tolerance to CII, are crucial for the suppression of CIA and could be exploited for immunotherapy of autoimmune diseases.

     

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.

     

Host‐derived CD4+ T cells attenuate stem cell–mediated transfer of autoimmune arthritis in lethally irradiated C57BL/6.g7 mice

Objective

In the K/BxN mouse model of inflammatory arthritis, T cells carrying a transgenic T cell receptor initiate disease by helping B cells to produce arthritogenic anti–glucose‐6‐phosphate isomerase (anti‐GPI) autoantibodies. We found that lethally‐ irradiated lymphocyte‐deficient C57BL/6 (B6).g7 (I‐A^g7+) recombinase‐activating gene–deficient (Rag^−/−) mice reconstituted with K/BxN hematopoietic stem and progenitor cells exhibit arthritis by week 4. In contrast, healthy B6.g7 recipients of K/BxN hematopoietic stem and progenitor cells show only mild arthritis, with limited extent and duration. The objective of this study was to investigate the factors responsible for the attenuation of arthritis in B6.g7 recipients.

Methods

Antibody responses were measured by enzyme‐linked immunosorbent assay. Fluorescence‐activated cell sorting analyses were performed for testing chimerism, expression of markers of activation and suppression, tetramer binding, and intracellular cytokines in CD4+ T cells. Suppressive activity of CD4+ T cells was studied by adoptive transfer.

Results

Titers of anti‐GPI antibodies in reconstituted B6.g7 mice were ∼60‐fold lower than in reconstituted B6.g7 Rag^−/− mice. Examination of chimerism in the reconstituted B6.g7 mice showed that B cells and myeloid cells in these mice were donor derived, but CD4+ T cells were primarily host derived and enriched for cells expressing the conventional regulatory markers CD25 and FoxP3. Notably, CD4+CD25−FoxP3− T cells expressed markers of suppressive function (CD73 and folate receptor 4), and delayed disease after adoptive transfer. Activation of donor‐derived CD4+ T cells was reduced, and thymic deletion of these cells appeared increased.

Conclusion

Despite myeloablation, host CD4+ T cells having a regulatory phenotype emerge in these mice and attenuate autoimmunity.

     

Presence of a population of CD20+,CD38− B lymphocytes with defective proliferative responsiveness in the synovial compartment of patients with rheumatoid arthritis

Objective

To provide a comprehensive understanding of the humoral immune response that takes place at the site of inflammation in rheumatoid arthritis (RA), we studied the functional properties of synovial B cells. In particular, the response to various modes of mitogen stimulation was investigated.

Methods

Purified synovial fluid (SF) B cells were cultured in the presence of CD40 ligand (CD40L)–expressing fibroblasts and cytokines, activated T cells, or phorbol myristate acetate (PMA)/ionomycin. Proliferation was determined by ³H‐thymidine incorporation. Release of intracellular calcium was studied by flow cytometry.

Results

The inflamed joints of RA patients contained a population of CD20+,CD38− B cells with dramatically impaired mitogen responsiveness. Although the Ig‐producing capacity was intact, these cells failed to proliferate in response to (a) CD40 in the presence of interleukin‐2 (IL‐2) and IL‐10, (b) activated T cells, or (c) stimulation via the B cell receptor. Moreover, SF CD20+,CD38− B cells revealed a defective B cell receptor–induced Ca²⁺ influx, reminiscent of anergic B cells. Release of intracellular Ca²⁺ by ionomycin in the presence of the protein kinase C activator PMA did not restore the proliferative capacity. These findings indicate blockades in the proximal and distal intermediates involved in mitogen signaling.

Conclusion

SF CD20+,CD38− B cells have functionally impaired proliferative responsiveness. The capacity of these cells to respond to activation by the production of Ig supports the notion that these cells might serve as Ig‐producing effector cells and, as such, play a role in the pathophysiology of RA.

     

CD4+CD28− T cell expansion in granulomatosis with polyangiitis (Wegener's) is driven by latent cytomegalovirus infection and is associated with an increased risk of infection and mortality

Objective

Expanded populations of CD4+CD28− T cells with a cytotoxic phenotype have been repeatedly reported in patients with granulomatosis with polyangiitis (Wegener's) (GPA). In healthy individuals expansion of this T cell population follows cytomegalovirus (CMV) infection. We undertook this study to investigate whether CMV infection may be responsible for driving the expansion of CD4+CD28− T cells in GPA patients and how this might relate to clinical features.

Methods

Forty‐eight GPA patients and 38 age‐matched healthy donors were included in the study. CMV‐specific IgG in serum was detected by enzyme‐linked immunosorbent assay. Flow cytometric analysis was used to study T cell populations and phenotype. The presence of CMV in renal biopsy tissue from GPA patients was investigated by immunohistochemistry and polymerase chain reaction (PCR). Clinical information was obtained from patient records.

Results

Populations of CD4+CD28− T cells were only expanded in CMV‐seropositive GPA patients and controls. In CMV‐seropositive GPA patients we observed negative correlations between the percentages of CD4+CD28− T cells and both the percentage of naive T cells and the glomerular filtration rate at presentation. There was a significant association between the percentage of CD4+CD28− T cells and risk of infection and mortality. CMV could not be detected in renal tissue by PCR or immunohistochemistry. CMV seropositivity itself was not a risk factor for infection in a cohort of 182 patients with antineutrophil cytoplasmic antibody–associated vasculitis who had been recruited into clinical trials performed by the European Vasculitis Study Group.

Conclusion

The expansion of CD4+CD28− T cells in GPA patients is associated with CMV infection and leads to a reduction in the number of naive T cells in peripheral blood. Patients with expanded CD4+CD28− T cells have significantly increased mortality and risk of infection.

     

ANTI‐CD3 therapy expands the numbers of CD4+ and CD8+ treg cells and induces sustained amelioration of collagen‐induced arthritis

Objective

To assess the therapeutic potential of anti‐CD3 monoclonal antibodies (mAb) for rheumatoid arthritis, using collagen‐induced arthritis as an animal model.

Methods

Arthritis was induced in DBA/1 mice by immunization with type II collagen. After disease onset, a single injection of anti‐CD3 mAb (20 μg/mouse) was administered, and arthritis severity was monitored over a 10‐day period.

Results

Anti‐CD3 mAb treatment resulted in a sustained reduction in disease activity, which was associated with an increase in the proportion of naturally occurring CD4+CD25+FoxP3+ regulatory T (Treg) cells and the generation of a population of CD8+CD25+FoxP3+ Treg cells. Anti‐CD3 mAb treatment did not alter the capacity of CD4+ Treg cells to suppress effector T cell proliferation and interferon‐γ (IFNγ) production in vitro. However, CD4+ Treg cells from both anti‐CD3 mAb–treated and control mice were unable to suppress interleukin‐17 (IL‐17) production. In contrast, CD8+ Treg cells induced by anti‐CD3 therapy suppressed IL‐17 production as well as CD4+ T cell proliferation and IFNγ production.

Conclusion

These results show that anti‐CD3 mAb treatment has important therapeutic potential for rheumatoid arthritis and has the capacity to generate antiarthritic CD8+ Treg cells and expand the relative numbers of CD4+ Treg cells.