Tobias Alexander

Depletion of autoreactive immunologic memory followed by autologous hematopoietic stem cell transplantation in patients with refractory SLE induces long-term remission through de novo generation of a juvenile and tolerant immune system

Clinical trials have indicated that immunoablation followed by autologous hematopoietic stem cell transplantation (ASCT) has the potential to induce clinical remission in patients with refractory systemic lupus erythematosus (SLE), but the mechanisms have remained unclear. We now report the results of a single-center prospective study of long-term immune reconstitution after ASCT in 7 patients with SLE. The clinical remissions observed in these patients are accompanied by the depletion of autoreactive immunologic memory, reflected by the disappearance of pathogenic anti-double-stranded DNA (dsDNA) antibodies and protective antibodies in serum and a fundamental resetting of the adaptive immune system. The latter comprises recurrence of CD31(+)CD45RA(+)CD4(+) T cells (recent thymic emigrants) with a doubling in absolute numbers compared with age-matched healthy controls at the 3-year follow-up (P = .016), the regeneration of thymic-derived FoxP3(+) regulatory T cells, and normalization of peripheral T-cell receptor (TCR) repertoire usage. Likewise, responders exhibited normalization of the previously disturbed B-cell homeostasis with numeric recovery of the naive B-cell compartment within 1 year after ASCT. These data are the first to demonstrate that both depletion of the autoreactive immunologic memory and a profound resetting of the adaptive immune system are required to reestablish self-tolerance in SLE.

Human Chorionic Gonadotropin Attracts Regulatory T Cells into the Fetal-Maternal Interface during Early Human Pregnancy

Regulatory T cells (Treg) expand during pregnancy and are present at the fetal-maternal interface at very early stages in pregnancy. The migration mechanisms of Treg to the pregnant uterus are still unclear. Human chorionic gonadotropin (hCG) is secreted by the blastocyst immediately after fertilization and has chemoattractant properties. Therefore, we sought to analyze whether hCG secreted by early trophoblasts attracts Treg to the uterus and hence contributes to maternal tolerance toward the fetus. Decidua and placenta tissue samples from patients having spontaneous abortions or ectopic pregnancies were employed to evaluate Treg and hCG levels. Age-matched samples from normal pregnant women served as controls. We further performed in vitro studies with primary first trimester trophoblast cells and a choriocarcinoma cell line (JEG-3) aiming to evaluate the ability of secreted hCG to attract Treg. Patients having miscarriages or ectopic pregnancy presented significantly decreased hCG mRNA and protein levels associated with decreased Foxp3, neuropilin-1, IL-10, and TGF-β mRNA levels as compared with normal pregnant women. Using migration assays we demonstrated that Treg were attracted by hCG-producing trophoblasts or choriocarcinoma cells. Treg migration toward cells transfected with hCG expression vectors confirmed the chemoattractant ability of hCG. Our data clearly show that hCG produced by trophoblasts attracts Treg to the fetal-maternal interface. High hCG levels at very early pregnancy stages ensure Treg to migrate to the site of contact between paternal Ags and maternal immune cells and to orchestrate immune tolerance toward the fetus.

The proteasome inhibitior bortezomib depletes plasma cells and ameliorates clinical manifestations of refractory systemic lupus erythematosus

Objectives To investigate whether bortezomib, a proteasome inhibitor approved for treatment of multiple myeloma, induces clinically relevant plasma cell (PC) depletion in patients with active, refractory systemic lupus erythematosus (SLE). Methods Twelve patients received a median of two (range 1–4) 21-day cycles of intravenous bortezomib (1.3 mg/m2) with the coadministration of dexamethasone (20 mg) for active SLE. Disease activity was assessed using the SLEDAI-2K score. Serum concentrations of anti–double-stranded DNA (anti-dsDNA) and vaccine-induced protective antibodies were monitored. Flow cytometry was performed to analyse peripheral blood B-cells, PCs and Siglec-1 expression on monocytes as surrogate marker for type-I interferon (IFN) activity. Results Upon proteasome inhibition, disease activity significantly declined and remained stable for 6 months on maintenance therapies. Nineteen treatment-emergent adverse events occurred and, although mostly mild to moderate, resulted in treatment discontinuation in seven patients. Serum antibody levels significantly declined, with greater reductions in anti-dsDNA (∼60%) than vaccine-induced protective antibody titres (∼30%). Bortezomib significantly reduced the numbers of peripheral blood and bone marrow PCs (∼50%), but their numbers increased between cycles. Siglec-1 expression on monocytes significantly declined. Conclusions These findings identify proteasome inhibitors as a putative therapeutic option for patients with refractory SLE by targeting PCs and type-I IFN activity, but our results must be confirmed in controlled trials.

Secondary autoimmune diseases occurring after HSCT for an autoimmune disease : a retrospective study of the EBMT Autoimmune Disease Working Party

To specify the incidence and risk factors for secondary autoimmune diseases (ADs) after HSCT for a primary AD, we retrospectively analyzed AD patients treated by HSCT reported to EBMT from 1995 to 2009 with at least 1 secondary AD (cases) and those without (controls). After autologous HSCT, 29 of 347 patients developed at least 1 secondary AD within 21.9 (0.6-49) months and after allogeneic HSCT, 3 of 16 patients. The observed secondary ADs included: autoimmune hemolytic anemia (n = 3), acquired hemophilia (n = 3), autoimmune thrombocytopenia (n = 3), antiphospholipid syndrome (n = 2), thyroiditis (n = 12), blocking thyroid-stimulating hormone receptor antibody (n = 1), Graves disease (n = 2), myasthenia gravis (n = 1), rheumatoid arthritis (n = 2), sarcoidosis (n = 2), vasculitis (n = 1), psoriasis (n = 1), and psoriatic arthritis (n = 1). After autologous HSCT for primary AD, the cumulative incidence of secondary AD was 9.8% ± 2% at 5 years. Lupus erythematosus as primary AD, and antithymocyte globulin use plus CD34(+) graft selection were important risk factors for secondary AD by multivariate analysis. With a median follow-up of 6.2 (0.54-11) years after autologous HSCT, 26 of 29 patients with secondary AD were alive, 2 died during their secondary AD (antiphospholipid syndrome, hemophilia), and 1 death was HSCT-related. This European multicenter study underlines the need for careful management and follow-up for secondary AD after HSCT.

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.

Foxp3+ Helios+ regulatory T cells are expanded in active systemic lupus erythematosus

Objectives Recent data debate the suitability of Helios, an Ikaros family member, as a marker for thymic-derived regulatory T cells (Treg). Nevertheless, Foxp3+ Helios+ Treg may be of particular relevance in mediating immune tolerance in chronic autoimmunity, such as systemic lupus erythematosus (SLE), as they possess enhanced suppressive function, compared to Foxp3+ Helios− Treg. Methods Multicolour flow cytometry was performed to analyse Foxp3 and Helios expression in peripheral blood CD4 T cells from SLE patients, compared to healthy controls (HC) and systemic sclerosis (SSc) and rheumatoid arthritis (RA) patients. Cytokine production, chemokine receptor expression for CXCR3 and CCR4, basal signal transducer and activator of transcription 5 (STAT5)a phosphorylation levels and T-cell receptor (TCR) Vβ repertoire were analysed by flow cytometry, and the methylation status of the Foxp3 locus (Treg-specific demethylated region, TSDR) by real-time PCR. Results Frequencies of Foxp3+ Helios+ Treg, unlike Foxp3+ Helios− T cells, were significantly increased in SLE patients and positively correlated with disease activity, whereas they were unaltered in SSc and RA patients. Compared to HC, Foxp3+ Helios+ Treg in SLE predominantly displayed a CD45RA−/CD31−/FoxP3low memory phenotype with increased Ki-67 expression, enhanced basal pSTAT5a levels and a restricted TCR repertoire. Nonetheless, similar to HC, Foxp3+ Helios+ Treg in SLE lacked effector cytokine production, possessed a highly demethylated TSDR and expressed comparable levels of CXCR3 and CCR4. Conclusions Our data suggest that Helios-expressing Foxp3+ Treg with functional suppressive capacity and migratory potential into inflamed tissues are expanded in active SLE, presumably through γ-chain signalling cytokines and TCR stimulation, to compensate for autoreactive effector responses.

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 …

A unique population of IgG-expressing plasma cells lacking CD19 is enriched in human bone marrow.

Specific serum antibodies mediating humoral immunity and autoimmunity are provided by mature plasma cells (PC) residing in the bone marrow (BM), yet their dynamics and composition are largely unclear. We here characterize distinct subsets of human PC differing by CD19 expression. Unlike CD19(+) PC, CD19(-) PC were restricted to BM, expressed predominantly IgG, and they carried a prosurvival, distinctly mature phenotype, that is, HLA-DR(low)Ki-67(-)CD95(low)CD28(+)CD56(+/-), with increased BCL2 and they resisted their mobilization from the BM after systemic vaccination. Fewer mutations within immunoglobulin VH rearrangements of CD19(-) BMPC may indicate their differentiation in early life. Their resistance to in vivo B-cell depletion, that is, their independency from supply with new plasmablasts, is consistent with long-term stability of this PC subset in the BM. Moreover, CD19(-) PC were detectable in chronically inflamed tissues and secreted autoantibodies. We propose a multilayer model of PC memory in which CD19(+) and CD19(-) PC represent dynamic and static components, respectively, permitting both adaptation and stability of humoral immune protection.

Autologous haematopoietic stem cell transplantation for systemic lupus erythematosus: Data from the European Group for Blood and Marrow Transplantation registry

Objectives Patients with systemic lupus erythematosus (SLE) refractory to conventional immunosuppression suffer substantial morbidity and mortality due to active disease and treatment toxicity. Immunoablation followed by autologous stem cell transplantation (ASCT) is a novel therapeutic strategy that potentially offers new hope to these patients. Methods This retrospective survey reviews the efficacy and safety of ASCT in 28 SLE patients from eight centres reported to the European Group for Blood and Marrow Transplantation (EBMT) registry between 2001 and 2008. Results Median disease duration before ASCT was 52 (nine to 396) months, 25/28 SLE patients (89%) were female, age 29 (16–48) years. At the time of ASCT, eight (one to 11) American College of Rheumatology (ACR) diagnostic criteria for SLE were present and 17 (60%) patients had nephritis. Peripheral blood stem cells were mobilized with cyclophosphamide and granulocyte-colony stimulating factor in 93% of patients, and ex vivo CD34 stem cell selection was performed in 36%. Conditioning regimens were employed with either low (n = 10) or intermediate (18) intensities. With a median follow-up of 38 (one to 110) months after ASCT, the five-year overall survival was 81 ± 8%, disease-free survival was 29 ± 9%, relapse incidence (RI) was 56 ± 11% and non-relapse mortality was 15 ± 7%. Graft manipulation by CD34+ selection was associated with a lower RI (p = 0.001) on univariate analysis. There were five deaths within two years after ASCT: three caused by infection, one by secondary autoimmune disease and one by progressive SLE. Conclusions Our data further support the concept of immunoablation and ASCT to re-induce long-term clinical and serologic remissions in refractory SLE patients even in the absence of maintenance therapy. This study also suggests a beneficial effect of ex vivo graft manipulation on prevention of relapses post-transplantation in SLE.

The Immunoregulator Soluble TACI Is Released by ADAM10 and Reflects B Cell Activation in Autoimmunity

BAFF and a proliferation-inducing ligand (APRIL), which control B cell homeostasis, are therapeutic targets in autoimmune diseases. TACI-Fc (atacicept), a soluble fusion protein containing the extracellular domain of the BAFF–APRIL receptor TACI, was applied in clinical trials. However, disease activity in multiple sclerosis unexpectedly increased, whereas in systemic lupus erythematosus, atacicept was beneficial. In this study, we show that an endogenous soluble TACI (sTACI) exists in vivo. TACI proteolysis involved shedding by a disintegrin and metalloproteinase 10 releasing sTACI from activated B cells. The membrane-bound stub was subsequently cleaved by γ-secretase reducing ligand-independent signaling of the remaining C-terminal fragment. The shed ectodomain assembled ligand independently in a homotypic way. It functioned as a decoy receptor inhibiting BAFF- and APRIL-mediated B cell survival and NF-κB activation. We determined sTACI levels in autoimmune diseases with established hyperactivation of the BAFF–APRIL system. sTACI levels were elevated both in the cerebrospinal fluid of the brain-restricted autoimmune disease multiple sclerosis correlating with intrathecal IgG production, as well as in the serum of the systemic autoimmune disease systemic lupus erythematosus correlating with disease activity. Together, we show that TACI is sequentially processed by a disintegrin and metalloproteinase 10 and γ-secretase. The released sTACI is an immunoregulator that shares decoy functions with atacicept. It reflects systemic and compartmentalized B cell accumulation and activation.