Jens Y. Humrich

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.

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, approximately 60% of them were T cells, and the CXCR3+ cells colocalized with CXCL10-producing cells. In biopsy tissues from SLE patients with acute nephritis, approximately 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.

Autoregulation of Th1-mediated inflammation by twist1

The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor κB (NF-κB)–dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-κB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohns disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-γ, IL-2, and tumor necrosis factor-α, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.

Low-dose interleukin-2 selectively corrects regulatory T cell defects in patients with systemic lupus erythematosus

Objectives Defects in regulatory T cell (Treg) biology have been associated with human systemic autoimmune diseases, such as systemic lupus erythematosus (SLE). However, the origin of such Treg defects and their significance in the pathogenesis and treatment of SLE are still poorly understood. Methods Peripheral blood mononuclear cells (PBMC) from 61 patients with SLE and 52 healthy donors and in vitro IL-2 stimulated PBMC were characterised by multicolour flow cytometry. Five patients with refractory SLE were treated daily with subcutaneous injections of 1.5 million IU of human IL-2 (aldesleukin) for five consecutive days, and PBMC were analysed by flow cytometry. Results Patients with SLE develop a progressive homeostatic dysbalance between Treg and conventional CD4+ T cells in correlation with disease activity and in parallel display a substantial reduction of CD25 expression on Treg. These Treg defects resemble hallmarks of IL-2 deficiency and lead to a markedly reduced availability of functionally and metabolically active Treg. In vitro experiments revealed that lack of IL-2 production by CD4+ T cells accounts for the loss of CD25 expression in SLE Treg, which could be selectively reversed by stimulation with low doses of IL-2. Accordingly, treatment of patients with SLE with a low-dose IL-2 regimen selectively corrected Treg defects also in vivo and strongly expanded the Treg population. Conclusions Treg defects in patients with SLE are associated with IL-2 deficiency, and can be corrected with low doses of IL-2. The restoration of endogenous mechanisms of immune tolerance by low-dose IL-2 therapy, thus, proposes a selective biological treatment strategy, which directly addresses the pathophysiology in SLE.

Rapid induction of clinical remission by low-dose interleukin-2 in a patient with refractory SLE

Interleukin-2 (IL-2) is crucial for the growth and survival of regulatory T cells (Treg) and, thus, for the control of autoimmunity.1 ,2 In previous studies, we have proven a causal relationship between an acquired IL-2-deficiency, defects in Treg biology and the development of systemic lupus erythematosus (SLE).3 Accordingly, we showed that compensation of IL-2 deficiency by IL-2 therapy corrects associated Treg defects and ameliorates already established disease in lupus-prone mice.3 In line with this, two independent clinical studies showed recently that low-dose IL-2 induced expansion of the Treg pool and reduced clinical symptoms in two different immunological diseases without known IL-2 deficiency.4 ,5 These promising clinical findings in conjunction with preclinical data3 ,6 ,7 provide strong rationales for an IL-2-based immunotherapy of SLE in order to restore Treg activity and, thus, to re-establish endogenous mechanisms of tolerance that can counteract autoimmunity. Here we report a rapid and robust reduction of disease activity in parallel with a remarkable expansion of the Treg population by low-dose IL-2 therapy in one patient with a long-term history of SLE and increased disease activity refractory …

The highly attenuated vaccinia virus strain modified virus Ankara induces apoptosis in melanoma cells and allows bystander dendritic cells to generate a potent anti‐tumoral immunity

Vaccinia virus (VV) has been tested as oncolytic virus against malignant melanoma in clinical trials for more than 40 years. Until now, mainly strains comparable to viral strains used for smallpox vaccination have been probed for anti‐tumoral therapy. We have shown recently that the wild‐type strain Western Reserve (WR) can interfere with crucial functions of monocyte‐derived dendritic cells (DCs). Our aim was to examine whether viral immune evasion mechanisms might be responsible for the ineffectiveness of WR‐based vaccination strategies and whether the highly attenuated strain modified virus Ankara (MVA) differs from WR with respect to its possible immunostimulatory capacity after intratumoral injection. Using in vitro experiments, we compared the effect of both strains on melanoma cells and on local bystander DCs. We found that both VV‐strains infected melanoma cells efficiently and caused disintegration of the actin cytoskeleton, as shown by fluorescence microscopy. In addition, both VV‐strains caused apoptotic cell death in melanoma cells after infection. In contrast to MVA, WR underwent a complete viral replication cycle in melanoma cells. Bystander DCs were consecutively infected by newly generated WR virions and lost their capacity to induce allogeneic T cell proliferation. DCs in contact with MVA‐infected melanoma cells retained their capacity to induce T cell proliferation. Immature DCs were capable of phagocytosing MVA‐infected melanoma cells. Priming of autologous CD8+ T cells by DCs that had phagocytosed MVA‐infected, MelanA positive melanoma cells resulted in the induction of T cell clones specifically reactive against the model antigen MelanA as shown by enzyme‐linked immunospot (ELISPOT) analysis. We conclude that the clinical trials with oncolytic wild‐type VV failed probably because of suppression of bystander DCs and consecutive suppression of T cell‐mediated anti‐melanoma immunity. The attenuated VV‐strain MVA facilitates the generation of tumour associated antigen (TAA)‐specific T cell response as it is oncolytic for melanoma cells, but non‐toxic for DC, and should be a promising candidate for intralesional metastatic melanoma therapy.

Mature monocyte‐derived dendritic cells respond more strongly to CCL19 than to CXCL12: consequences for directional migration

The chemokine receptor CCR7 is crucial for migration of mature dendritic cells (DC) directed toward secondary lymphoid organs; however, there is little knowledge about the function of the homeostatic chemokine receptor CXCR4 in DC and its contribution to directional migration of DC during inflammation. By comparing the impact of chemokine receptor engagement on mature DC we found that the CCR7 ligand CCL19 holds a stronger chemotactic potency than the CXCR4 ligand CXCL12. Moreover, CCL19 elicited rapid, steep and long‐lasting mobilization of intracellular calcium in individual cells and induced intense phosphorylation of extracellular signal‐regulated kinase 1/2 and protein kinase B, while the intracellular signals elicited by CXCL12 were in part distinct and significantly weaker. Analysis of chemokine receptor expression revealed that although CCR7 and CXCR4 were expressed by a similar percentage of DC, the mean fluorescence intensity of CCR7 was up to six times higher, suggesting a higher receptor density. Based on these correlations we propose that the type of chemokine signal in conjunction with the expression and functional activity of the respective chemokine receptor is also determining the migration rate and potency of a chemotactic response in mature DC. In conclusion, our data support the fundamental role of CCR7 for rapidly guiding DC toward secondary lymphoid organs at an extra‐ and intracellular molecular level and on the contrary render CXCR4 a weaker contributor to directional migration of DC during inflammation.

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.