Reinmar Undeutsch

Homeostatic imbalance of regulatory and effector T cells due to IL-2 deprivation amplifies murine lupus.

The origins and consequences of a regulatory T cell (Treg) disorder in systemic lupus erythematosus (SLE) are poorly understood. In the (NZBxNZW) F1 mouse model of lupus, we found that CD4+Foxp3+ Treg failed to maintain a competitive pool size in the peripheral lymphoid organs resulting in a progressive homeostatic imbalance of CD4+Foxp3+ Treg and CD4+Foxp3− conventional T cells (Tcon). In addition, Treg acquired phenotypic changes that are reminiscent of IL-2 deficiency concomitantly to a progressive decline in IL-2-producing Tcon and an increase in activated, IFN-γ-producing effector Tcon. Nonetheless, Treg from lupus-prone mice were functionally intact and capable to influence the course of disease. Systemic reduction of IL-2 levels early in disease promoted Tcon hyperactivity, induced the imbalance of Treg and effector Tcon, and strongly accelerated disease progression. In contrast, administration of IL-2 partially restored the balance of Treg and effector Tcon by promoting the homeostatic proliferation of endogenous Treg and impeded the progression of established disease. Thus, an acquired and self-amplifying disruption of the Treg-IL-2 axis contributed essentially to Tcon hyperactivity and the development of murine lupus. The reversibility of this homeostatic Treg disorder provides promising approaches for the treatment of SLE.

Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis

IntroductionVasculopathy, inflammatory fibrosis and functional autoantibodies (Abs) are major manifestations of systemic sclerosis (SSc). Abs directed against the angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) are associated with characteristic disease features including vascular, inflammatory, and fibrotic complications indicating their role in SSc pathogenesis. Therefore, the impact of anti-AT1R and anti-ETAR Abs on initiation of inflammation and fibrosis was analyzed.MethodsAnti-AT1R and anti-ETAR Ab-positive immunoglobulin G (IgG) from SSc patients (SSc-IgG) was used for experiments. Healthy donor IgG served as a normal control, and AT1R and ETAR activation was inhibited by antagonists. Protein expression was measured with ELISA, mRNA expression with real time-PCR, endothelial repair with a scratch assay, and collagen expression with immunocytochemistry. Transendothelial neutrophil migration was measured with a culture insert system, and neutrophil ROS activation with immunofluorescence. Neutrophils in bronchoalveolar lavage fluids (BALFs) were analyzed microscopically after passive transfer of SSc-IgG or NC-IgG into naïve C57BL/6J mice. KC plasma levels were quantified by a suspension array system. Histologic analyses were performed by using light microscopy.ResultsAnti-AT1R and anti-ETAR Ab-positive SSc-IgG induced activation of human microvascular endothelial cells (HMEC-1). Elevated protein and mRNA levels of the proinflammatory chemokine interleukin-8 (IL-8, CXCL8) and elevated mRNA levels of the vascular cell adhesion molecule-1 (VCAM-1) were induced in HMEC-1. Furthermore, activation of HMEC-1 with SSc-IgG increased neutrophil migration through an endothelial cell layer and activation of reactive oxygen species (ROS). SSc-IgG decreased HMEC-1 wound repair and induced type I collagen production in healthy donor skin fibroblasts. Effects of migration, wound repair, and collagen expression were dependent on the Ab-levels. Passive transfer of anti-AT1R and anti-ETAR Ab-positive SSc-IgG into naïve C57BL/6J mice increased neutrophil BALF counts. In parallel, increased levels of the murine functional IL-8 homologue, chemokine KC, were found in the plasma of SSc-IgG-treated mice as well as structural alterations of the lungs.ConclusionsWe conclude that angiotensin and endothelin-receptor activation via anti-AT1R and anti-ETAR Abs mediate pathogenic effects, indicating their contribution to pathogenesis of SSc. Therefore, anti-AT1R and anti-ETAR Abs could provide novel targets for therapeutic intervention in the treatment of SSc.

CD4+Foxp3+ regulatory T cells prolong drug-induced disease remission in (NZBxNZW) F1 lupus mice

IntroductionThe ability to ameliorate murine lupus renders regulatory T cells (Treg) a promising tool for the treatment of systemic lupus erythematosus (SLE). In consideration to the clinical translation of a Treg-based immunotherapy of SLE, we explored the potential of CD4+Foxp3+ Treg to maintain disease remission after induction of remission with an established cyclophosphamide (CTX) regimen in lupus-prone (NZBxNZW) F1 mice. As a prerequisite for this combined therapy, we also investigated the impact of CTX on the biology of endogenous Treg and conventional CD4+ T cells (Tcon).MethodsRemission of disease was induced in diseased (NZBxNZW) F1 mice with an established CTX regimen consisting of a single dose of glucocorticosteroids followed by five day course with daily injections of CTX. Five days after the last CTX injection, differing amounts of purified CD4+Foxp3+CD25+ Treg were adoptively transferred and clinical parameters, autoantibody titers, the survival and changes in peripheral blood lymphocyte subsets were determined at different time points during the study. The influence of CTX on the numbers, frequencies and proliferation of endogenous Treg and Tcon was analyzed in lymphoid organs by flow cytometry.ResultsApart from abrogating the proliferation of Tcon, we found that treatment with CTX induced also a significant inhibition of Treg proliferation and a decline in Treg numbers in lymphoid organs. Additional adoptive transfer of 1.5 × 106 purified Treg after the CTX regimen significantly increased the survival and prolonged the interval of remission by approximately five weeks compared to mice that received only the CTX regimen. The additional clinical amelioration was associated with an increase in the Treg frequency in the peripheral blood indicating a compensation of CTX-induced Treg deficiency by the Treg transfer.ConclusionsTreg were capable to prolong the interval of remission induced by conventional cytostatic drugs. This study provides valuable information and a first proof-of-concept for the feasibility of a Treg-based immunotherapy in the maintenance of disease remission in SLE.

Intravenous injection of a D1 protein of the Smith proteins postpones murine lupus and induces type 1 regulatory T cells

T cells that recognize nucleoproteins are required for the production of anti-dsDNA Abs involved in lupus development. SmD183–119 (a D1 protein of the Smith (Sm) proteins, part of small nuclear ribonucleoprotein) was recently shown to provide T cell help to anti-dsDNA Abs in the NZB/NZW model of lupus. Using this model in the present study, we showed that high dose tolerance to SmD1 (600–1000 μg i.v. of SmD183–119 peptide/mo) delays the production of autoantibodies, postpones the onset of lupus nephritis as confirmed by histology, and prolongs survival. Tolerance to SmD183–119 was adoptively transferred by CD90+ T cells, which also reduce T cell help for autoreactive B cells in vitro. One week after SmD183–119 tolerance induction in prenephritic mice, we detected cytokine changes in cultures of CD90+ T and B220+ B cells with decreased IFN-γ and IL-4 expression and an increase in TGFβ. Increased frequencies of regulatory IFN-γ+ and IL10+ CD4+ T cells were later detected. Such regulatory IL-10+/IFN-γ+ type 1 regulatory T cells prevented autoantibody generation and anti-CD3-induced proliferation of naive T cells. In conclusion, these results indicate that SmD183–119 peptide may play a dominant role in the activation of helper and regulatory T cells that influence autoantibody generation and murine lupus.

Urinary CD4 T cells identify SLE patients with proliferative lupus nephritis and can be used to monitor treatment response

Objectives Proliferative lupus nephritis (LN) is one of the major concerns in the treatment of systemic lupus erythematosus (SLE). Here we evaluate urinary CD4 T cells as a biomarker of active LN and indicator of treatment response. Methods Urinary CD3CD4 T cells were quantified using flow cytometry in 186 urine samples from 147 patients with SLE. Fourteen patients were monitored as follow-up. Thirty-one patients with other nephropathies and 20 healthy volunteers were included as controls. Results In SLE, urinary CD4 T cell counts ≥800/100 ml were observed exclusively in patients with active LN. Receiver operator characteristic analysis documented clear separation of SLE patients with active and non-active LN (area under the curve 0.9969). All patients with up-to-date kidney biopsy results showing proliferative LN had high urinary CD4 T cell numbers. In patients monitored under therapy, normalisation of urinary CD4 T cell counts indicated lower disease activity and better renal function. In contrast, patients with persistence of, or increase in, urinary T cells displayed worse outcomes. Conclusions Urinary CD4 T cells are a highly sensitive and specific marker for detecting proliferative LN in patients with SLE. Furthermore, monitoring urinary CD4 T cells may help to identify treatment responders and treatment failure and enable patient-tailored therapy in the future.

Unmasking of autoreactive CD4 T cells by depletion of CD25 regulatory T cells in systemic lupus erythematosus

Objective Autoreactive CD4 T cells specific for nuclear peptide antigens play an important role in tolerance breakdown during the course of systemic lupus erythematosus (SLE). However, reliable detection of these cells is limited due to their low frequency in peripheral blood. The authors assess autoreactive CD4 T cells in a representative SLE collective (n=38) by flow cytometry and study the influence of regulatory T cells (Treg) on their antigenic challenge. Methods CD4 T-cell responses were determined according to intracellular CD154 expression induced after 6-h short-term in-vitro stimulation with the SLE-associated autoantigen SmD1(83-119). To clarify the influence of Treg on the activation of autoreactive CD4 T cells, CD25 Treg were depleted by magnetic activated cell sorting before antigen-specific stimulation in selected experiments. Results In the presence of Treg, autoreactive CD4 T-cell responses to SmD1(83-119) were hardly observable. However, Treg removal significantly increased the frequency of detectable SmD1(83-119)-specific CD4 T cells in SLE patients but not in healthy individuals. Consequently, by depleting Treg the percentage of SmD1(83-119)-reactive SLE patients increased from 18.2% to 63.6%. This unmasked autoreactivity of CD4 T cells correlated with the disease activity as determined by the SLE disease activity index (p=0.005*, r=0.779). Conclusions These data highlight the pivotal role of the balance between autoreactive CD4 T cells and CD25 Treg in the dynamic course of human SLE. Analysing CD154 expression in combination with a depletion of CD25 Treg, as shown here, may be of further use in approaching autoantigen-specific CD4 T cells in SLE and other autoimmune diseases.

Induction of pathogenic anti-dsDNA antibodies is controlled on the level of B cells in a non-lupus prone mouse strain.

The SmD183–119 peptide is a main target of autoantibodies and T cells in human and murine lupus, but its role in autoimmunity induction remains elusive. Therefore, female Balb/c mice and (NZW × Balb/c)F1 [CWF1] mice with identical MHC haplotype as lupus prone NZB/W mice were immunized with SmD183−119. Immunizations of CWF1 mice with SmD183–119, but not with the controls (irrelevant peptide, HEL peptide, or saline), induced anti-SmD183–119 and anti-dsDNA antibodies and proteinuria not present in Balb/c mice. DsDNA-specific plasma cell induction after SmD183–119 immunizations was confirmed by ELISPOT assays showing that the generation of dsDNA-specific antibody forming cells (AFC) was mainly driven by increased T-cell help. T-cell help for the generation of dsDNA-specific AFC was also present in saline-treated CWF1 mice but was controlled on the levels of B cells preventing autoimmunity.

SAT0165 Low-dose IL-2 therapy selectively expands regulatory T cells and suppresses murine lupus

Background Our previous studies in the (NZBxNZW) F1 model provide strong rationales for an IL-2 based immunotherapy of lupus in order to restore regulatory T cell (Treg) mediated tolerance that is impaired due to an acquired IL-2 deficiency (Humrich et al. 2010). However, although Treg appear to be the major target of IL-2 in vivo, because they constitutively express the high-affinity IL-2 receptor,other cells that potentially trigger autoimmunitycan also be activated by IL-2 in a dose dependent fashion. Objectives To determine an optimal dose and regimen for a therapy with IL-2 that induces a sufficient expansion of CD4+Foxp3+ Treg in vivo while only marginally affecting other cells, and that most efficiently influences active disease in the (NZBxNZW) F1 model for lupus. In addition, the underlying cellular mechanisms and side effects were studied. Methods Recombinant mouse IL-2 at different single doses (1, 5, 25, 50 ng/g body weight) was injected subcutaneously either into young or diseased (NZBxNZW) F1 mice every day for the duration of five days as induction therapy. After the induction phase, IL-2 injections were continued every 4 days until the end of the experiment. Control animals received an equal amount of PBS (carrier). Cells from lymphoid organs and peripheral blood were analyzed by flow cytometry at days 5, 7, 14 and 21. In addition survival and clinical parameters (weight, proteinuria, leukozyturia, autoantibodies) were analysed during IL-2 therapy of diseased mice for regimens with the single dosages of 5 and 25 ng/g body weight. Results We found that the low-dose IL-2 regimen with a single dose of 5ng/g body weight sufficiently promoted the expansion of CD4+Foxp3+Treg, while not or only marginally affecting CD4+ effector T cells and other cells. Although higher doses of IL-2 resulted in a more pronounced proliferation and expansion of Treg, this was accompanied by a considerable increase in CD44hi memory/effector CD4+ T cells and NK cells. Clinically, regimens with both 5ng/g and 25 ng/g were nearly equally sufficient to influence nephritis and to decrease mortality. In addition, we did not observe any signs of intolerability or other clinical side effects in the treated animals, even at higher dosages of 50ng/g body weight. Conclusions These studies provide the first evidence that low-dose IL-2 therapy selectively targets Treg and is clinically effective and also safe in murine lupus. This knowledge is very important for the future design of a clinical study with IL-2 in SLE. References Humrich, JY et al. (2010). Homeostatic imbalance of regulatory and effector T cells due to IL-2 deprivation amplifies murine lupus. Proc Natl Acad Sci USA 107, 204-9 Disclosure of Interest None Declared

SAT0182 Urinary T cells identify SLE patients with proliferative lupus nephritis and may be used to monitor treatment response

Background Lupus nephritis (LN) is one of the main concerns in the treatment of patients with SLE. However there are no good laboratory markers to non-invasively detect and monitor kidney inflammation in patients with LN. We and others have previously reported that urinary CD4 T cells may serve as a biomarker for acute LN, however these existing studies are limited by their relatively small sample size. Objectives To confirm that urinary CD4 T cells reflect lupus nephritis activity and may serve as a marker to monitor the local inflammation in the kidneys. Methods Urine samples from 118 patients with SLE and 10 healthy controls were analyzed using flowcytometry and correlated with the disease activity (SLEDAI). 24 SLE patients were analyzed at the same time point of receiving a kidney biopsy. Results In patients with renal involvement the amount of urinary CD4 T cells correlated closely with the disease activity (SLEDAI), while patients without renal involvement showed only marginal amounts of urinary T cells even during lupus flares. Healthy controls showed no urinary T cells. All patients with biopsy demonstrated proliferative LN showed highly elevated amounts of urinary CD4 T cells, while some patients with class V nephritis presented normal amounts of urinary T cells. Under treatment for acute LN one patient group quickly normalized the amount of urinary CD4 T cells while others showed a persistence of elevated urinary CD4 T cells counts. The latter patient group had a significantly higher disease activity 6 month after induction of treatment compared to the patients with normalized urinary CD4 T cells, indicating that urinary T cell count reflect treatment response. Conclusions Elevated numbers of urinary CD4 T cells reliably identify SLE patients with proliferative LN. Furthermore the amount of urinary CD4 T cells under treatment seems to reflect treatment response. We consequently propose that urinary CD4 T cells can be used to screen for proliferative LN and to monitor the LN activity under treatment. Disclosure of Interest None Declared

IL-2 therapy expands intrarenal FOXP3+ regulatory T cells and decreases the number of infiltrating CD4+T cell in murine lupus nephritis

Background and objectives Systemic lupus erythematosus (SLE) is an autoimmune disease characterised by an acquired IL-2 deficiency, which leads to a homeostatic imbalance between regulatory T cells (Treg) and effector T cells. Recently, the authors have shown that compensation of the IL-2 deficiency in diseased lupus mice by treatment with recombinant IL-2 (IL-2) ameliorates already established disease by promoting the homeostatic proliferation of regulatory T cell (Treg) in the lymphoid organs.1 The aim of this study was to investigate the impact of IL-2 therapy on intrarenal Foxp3+Treg and kidney infiltrating CD4+T cells in (NZBxNZW) F1 mouse model of lupus nephritis. Materials and methods (NZBxNZW) F1 mice with active nephritis were treated with recombinant IL-2 either over a short period of 24 h or over a long period of 20 days with PBS as control. Absolute numbers, phenotype and proliferation of kidney infiltrating CD4+T cells were determined by flow cytometry. Cellular infiltrates were also visualised by immunohistochemistry. Results Short term IL-2 treated (NZBxNZW) F1 mice resulted in an increase in numbers and frequency of CD4+Foxp3+Treg and strongly enhanced the proliferation of Foxp3+Treg compared to PBS treated control mice. In contrast, the long term IL-2 treatment over a period of 20 days did not result in a persistent expansion of the intrarenal Foxp3+Treg population. However, total numbers of kidney infiltrating CD4+Tcon were reduced in long term treated mice. In addition these CD4+Tcon showed reduced signs of cellular activation. Conclusions In this study, the authors found that short term IL-2 treatment is capable to expand the size of the intrarenal Treg pool. On the other hand, long term IL-2 treatment diminishes the numbers of kidney infiltrating CD4+Tcon. These findings underline the important role of intrarenal Treg for the suppression of kidney disease in lupus mice and may in part explain the delay of disease progression induced by treatment with IL-2. Furthermore, these data provide additional rationales for an IL-2 based immunotherapy of human disease.