AUTOIMMUNITY

Asian Pac J Allergy Immunol. 2019 Apr 23. doi: 10.12932/AP-211218-0465.[Epub ahead of print]...

The treatment of Lupus Nephritis (LN) is an unmet need in the management of patients with Systemic Lupus Erythematosus (SLE). We report two cases of women affected by Lupus Nephritis (LN) ISN/RNP Class IV with serological active disease, high disease activity and marked fatigue. In both cases, Mycophenolate mofetil (MMF), as induction therapy, was poorly tolerated because of gastrointestinal toxicity. Belimumab, together with low-doses of MMF, was effective as induction treatment leading to early achievement of complete renal response in these two selected cases of LN. We also report a literature review concerning the efficacy and safety of Belimumab in Lupus Nephritis. Further studies are needed to evaluate the use of Belimumab to manage the renal involvement in patients with Systemic Lupus Erythematosus, waiting for the results of ongoing randomized clinical trials.

Objectives Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterised by autoantibody production and widespread inflammation damaging many organs. Previous genome-wide association studies (GWASs) have revealed over 80 genetic determinants of SLE, but they collectively...

Objective Drug-induced lupus (DIL) is an idiosyncratic side effect of treatments in which symptoms overlap with those of systemic lupus erythematosus (SLE). The spectrum of DIL constantly evolves with that of the pharmacopoeia.

Full text KEYWORDS Antiphospholipid syndrome, antiphospholipid antibodies, thrombosis, pregnancy morbidity, catastrophic antiphospholipid syndrome, treatment INTRODUCTION  The antiphospholipid syndrome (APS) is defined by the occurrence of venous and/or arterial thrombotic events and/or pregnancy-related morbidity (≥ 3 unexplained consecutive spontaneous abortions < 10 weeks with exclusion of chromosomal causes, foetal death or severe pre-eclampsia before 34th week of gestation), combined with the presence of circulating antiphospholipid antibodies (aPL) and/or a lupus anticoagulant (LAC), see table 1.1 Formally, LAC is the result of aPL binding to plasma proteins, mainly β2-glycoprotein, that have affinity for the negatively-charged phospholipids; therefore, the pathologic auto-antibodies are not directed against phospholipids. In this paper, the term ‘aPL’ refers to both LAC and anticardiolipin (aCL)/anti-β2-glycoprotein-I (anti-β2GPI) antibodies, following clinical practice and literature. APS is considered a primary autoimmune disease, but is often diagnosed as being secondary to other auto-immune diseases; 30-40% of patients with systemic lupus erythematodes (SLE) also have APS, and APS can be diagnosed secondary to rheumatoid arthritis, systemic sclerosis, dermatomyositis or other autoimmune diseases.2 Large, controlled, randomised clinical trials for interventions in APS are limited, and the quality of current data is not sufficient to develop a structured guideline or assess evidence-based management strategies. However, clinically-relevant questions about diagnostic criteria and treatment of APS patients arise on a daily basis. We aim to provide clinicians with an expert consensus on the management of APS, with a focus on classification, diagnostics, risk stratification, and treatment.  METHODS A literature review was performed (KdL and ML) with the following Pubmed search terms: ‘antiphospholipid syndrome’, "Antiphospholipid Syndrome"[Mesh], ‘antiphospholipid antibodies’, "Antibodies, Antiphospholipid"[Mesh], ‘obstetric antiphospholipid syndrome’, ‘catastrophic antiphospholipid syndrome’, ‘laboratory diagnostics’, ‘Clinical Laboratory Techniques"[Mesh] ‘diagnosis’, "Diagnosis"[Mesh], ‘treatment’ and "Therapeutics"[Mesh]; papers focusing on diagnostics and/or treatment for APS were included. Non-English papers and papers published before 2000 were excluded. The literature search was performed on April 3rd, 2018; papers published after that date but with high impact according to the writing committee were included until September 1st, 2018. Statements on APS diagnostics and treatment were extracted from the papers selected, and a first draft of the consensus paper was written (KdL and ML). This draft was circulated amongst all authors and comments were collected. On June 6th, 2018, a first consensus meeting was held in Utrecht, The Netherlands. Based on the outcomes of this meeting, a second draft of the paper was written (JS, ML) and circulated again for comments. On November 5th, 2018, a second consensus meeting was held in Utrecht, The Netherlands. Degree of consensus on statements was assessed by a Delphi procedure and a final report was written afterwards (see table 3). Diagnosis, classification, risk stratification Diagnosis of APS No diagnostic criteria for APS exist. If a patient meets the classification criteria – developed for research purposes – for APS (e.g., a thrombotic event and/or pregnancy morbidity, combined with repeated presence of antiphospholipid antibodies (aPL); see table 1), most likely a clinical diagnosis of APS will be made, although thrombotic and pregnancy complications are not necessarily causally related to circulating aPL. In addition to the thrombotic or pregnancy-related events listed in the classification criteria, APS may be associated with a variety of non-criteria manifestations, such as superficial vein thrombosis, thrombocytopenia, renal microangiopathy, heart valve disease, livedo reticularis or racemosa, migraine, chorea, seizures and myelitis; see table 2. As a result, a clinical diagnosis of APS can be made in patients who do not fulfil the classification criteria.3 An even more complicating factor is the concept of seronegative APS, a term coined to include patients with clinical (criteria and non-criteria) features suggestive of APS, but who are persistently negative for aPL.4 The existence of seronegative APS is a point of international discussion among experts. If the suspicion of seronegative APS arises, we suggest referral to an expert centre. Catastrophic antiphospholipid syndrome (CAPS) is the most extreme APS variant that includes simultaneous multiple organ thrombosis and develops in a short period of time with a high mortality rate. Although strongly associated with the presence of LAC, no other laboratory or clinical determinants are known to be associated with CAPS.2 Risk stratification in APS Several assessment tools to risk-stratify patients have been proposed, mainly focusing on presence and levels of aPL and/or on clinical parameters. For aPL, the presence of LAC is the strongest risk factor for both arterial and venous thrombosis in APS.5,6 For aCL and anti-β2GPI, the association between (levels of) antibodies and thrombosis is less clear. It has been suggested that anti-β2GPIdependent LAC has a strong association with thrombotic risk.7 In several studies, it has been demonstrated that the risk of arterial and venous thrombosis increases with the number of positive tests for aPL, with the highest risks in patients with both LAC, aCL and anti-β2GPI antibodies, so-called ‘triple positive patients’.6,8 In clinical practice, risk stratification does not affect treatment decisions in most situations. The antiphospholipid score (aPL-S) has been developed to predict the risk of APS-related clinical events in patients with APS and other autoimmune diseases (such as SLE, rheumatoid arthritis, and Sjögren’s syndrome) based on the presence of aPL.9 The global APS score (GAPSS) is another clinical score, including both aPL and conventional cardiovascular risk factors, predicting the risk of thrombotic events in patients with SLE.10 The GAPSS has been validated in a cohort of APS patients, and a correlation between higher GAPSS values and recurrence of thrombotic events was observed.11 However, these scores are not sufficient to design treatment strategies for the individual patient. We consider triple positive patients to be at highest risk for recurrent thrombosis. Laboratory diagnostics in APS The classification criteria for APS indicate three different antibody subsets of aPL. For two of these, the antigen is well-defined: aCL antibodies recognize the plasma glycoprotein β2GPI in complex with the anionic phospholipid cardiolipin, and anti-β2GPI antibodies recognize the protein β2GPI in the absence of cardiolipin. Both antibody subtypes can be detected with quantitative solid phase assays in which the antigen is immobilized on a surface. The third aPL subtype, known as LAC, is detected with a functional assay: these antibodies manifest as phospholipid-dependent inhibitors of in vitro coagulation. They are detected with phospholipid sensitive coagulation assays. Although the exact antigen to which LAC are directed is currently unclear, there is ample evidence that antibodies against β2GPI as well as antibodies against prothrombin can induce the LAC phenomenon.12,13 The diagnosis of APS is made in cases of persistent presence of aPL (titre > 99th percentile), assessed in two separate samples taken with an arbitrarily defined interval of at least twelve weeks. This is an important distinction, as several aPL occur transiently in relation to viral and bacterial infections and are of uncertain clinical relevance.14 Moreover, since aPL is prevalent (1-5%) in the general population,15 aPL status should only be tested in patients considered at risk of having APS, such as those < 50 years of age, unprovoked arterial or venous thrombosis, thrombosis at an unusual site, recurrent thrombosis, and thrombotic/pregnancy complications with or without association with a systemic autoimmune disease.16-18 Unfortunately, gold standards for aPL detection are lacking, although aCL assays based on pooled human serum have been in use for over 20 years.19 Reports have been published of new standards based on human(ized) monoclonal antibodies against β2GPI and purified patient-derived polyclonal antibody preparations, but these are not yet available.20 No such standards exist for LAC-positive plasmas. For further harmonization of results between diagnostic laboratories, centres performing these tests participate in diagnostic surveys as part of laboratory accreditation. Measurement of anticardiolipin and anti-β2-glycoprotein I antibodies The classification criteria for APS specify that both immunoglobin G (IgG) and IgM class immunoglobulins against cardiolipin or β2GPI should be measured. Several commercial entities supply kits to measure these antibodies and many laboratories have developed their own solid phase assays. To minimize the effect of the lack of gold standards and the large number of assays in use for detection of aPL on assay standardization, guidance on assay characteristics has been provided by the Scientific Standardization Committee of the International Society on Thrombosis and Haemostasis.16 Detection of aCL and β2GPI antibodies can be performed in both trisodium citrate anticoagulated plasma and in serum. Since aCL antibodies associated with APS are β2GPI-dependent, diagnostic laboratories should use assays in which cardiolipin is saturated with human β2GPI. Samples should be considered positive when the value obtained in these assays exceeds the 99th percentile of the normal population, rather than antibody levels exceeding 40 arbitrary units as indicated in the classification criteria, as this appears to be more specific for APS.21 We recommend that the laboratory report mentions both a cut-off value (< 99th or > 99th percentile, e.g., negative or positive) and a continuous numeric value. Detection of lupus anticoagulant LAC are phospholipid-dependent coagulation inhibitors and are detected with sensitive coagulation assays in trisodium citrate anticoagulated plasma. Plasma samples should be double centrifuged to minimize contamination with platelets, as they are a major source of phospholipid and might therefore interfere with LAC detection.17,18 LAC can be detected with any phospholipid-dependent coagulation assay, however, no gold standard for LAC testing exists. For this reason, it is warranted to perform two tests based on a different assay principle for LAC detection, preferably a dilute Russell’s viper venom time (dRVVT) and a LAC-sensitive activated partial thromboplastin time (APTT).17 In order to be deemed LAC-positive, a sample should have a prolonged clotting time when a reagent with a low phospholipid content is used (screening test), which should correct when a reagent with a high phospholipid content is used (confirmatory test), indicating phospholipid-dependence of the prolongation. The presence of a coagulation inhibitor as the cause of the prolongation should be shown with a mixing test, in which patient plasma is mixed with an equal volume of pooled normal plasma. This will normalise any coagulation factor deficiencies that are present, and any remaining prolongation of the clotting time is therefore caused by an inhibitor. Cut-off values for LAC should be determined locally in each diagnostic laboratory, based on the 99th percentile of the local normal population or alternatively, on the mean + 2 standard deviation (SD) of the clotting time of the normal population. The strength of the LAC should be expressed as a ratio between screen and confirm clotting times, preferably normalized on the mean of the normal population, according to the following equation:   Samples are deemed positive for LAC when one or both tests for LAC detection (APTT and dRVVT-based tests) indicate the presence of LAC. The use of anticoagulant drugs interferes with detection of LAC, possibly resulting in false-positive test results. Samples for LAC-detection should therefore be collected before treatment with anticoagulants has started, or sufficiently long after cessation of treatment to minimize confounding effects. However, it should be avoided to use samples obtained in the acute phase after a thrombotic event or during infection, as this is associated with high Factor VIII levels, which might interfere with LAC detection by APTT-based assays. Although LAC can be determined in samples from patients receiving vitamin K antagonists when they are mixed with an equal volume of pooled normal plasma, the outcome of LAC tests in samples with an INR within therapeutic range (INR 2-3) should be interpreted with caution and measurement of LAC in samples with an international normalised ratio (INR) > 3 is not recommended. Mixing antagonists with normal plasma dilutes the titre of LAC and thus reduces the sensitivity of the assay. On the other hand, the mixing test may not completely correct the clotting time for samples in the high INR range and may lead to false-positive interpretation. Temporary discontinuation of vitamin K antagonists (or co-administration of vitamin K and continuing vitamin K antagonists) and bridging with low molecular weight heparin is possible. Unfractionated heparin, however, is incompatible with LAC testing, as is the use of direct oral anticoagulants, even at trough levels of factor Xa or thrombin inhibitors, with factor Xa inhibitors producing false-positive LAC.22 No alternatives exist for LAC detection in samples containing direct thrombin inhibitors. A possible means to detect LAC in samples containing rivaroxaban is the Taipan snake venom time/Ecarin clotting time combination, as these tests are insensitive to factor Xa-inhibitors.23 More studies on the specificity and sensitivity for LAC of this test combination are required before these tests will be widely adopted for LAC detection. Non-criteria aPL There are several reports on the association between various non-criteria aPL subtypes and thrombosis. Amongst these are IgA anti-β2GPI antibodies,24-26 antibodies against the phospholipid phosphatidylethanolamine (PE),27 antibodies against the complex between the phospholipid phosphatidylserine, and the coagulation factor prothrombin (aPS/PT).28 Currently, however, there is insufficient evidence of their clinical relevance to warrant routine detection of these antibodies. Treatment Venous thrombosis in APS A first venous thrombotic event (VTE; amongst others including deep vein thrombosis and pulmonary embolism, abdominal vein thrombosis, cerebral vein thrombosis) should be treated according to the current guidelines for treatment of VTE; no routine testing for aPL is indicated in the general population. In cases of recurrent thrombosis (both provoked and unprovoked), or patients with a pre-existing autoimmune disease (and in particular, SLE), additional testing for aPL should be performed. Low molecular weight heparin (LMWH) in therapeutic doses and subsequent vitamin K antagonists (VKA) are first-line treatments for a first or recurrent APS-related venous thrombotic event (VTE). Treatment with direct oral anticoagulants (DOACs; see separate section below) is not recommended. For APS patients with a first VTE, life-long anticoagulation is recommended. After treatment with LMWH in the acute phase, treatment will be switched to VKA, with an INR target range of 2.0-3.0 for venous events.29,30 High-intensity treatment with an INR ≥ 3.0 after a first VTE is not recommended.31,32 Arterial thrombosis in APS Optimal long-term treatment for arterial thrombosis (other than cerebral arterial thrombosis; see below) is still a matter of debate; either anti-platelet therapy such as aspirin or clopidogrel, VKA with an INR target range of 2.5-3.5 or combined therapy with VKA with an INR target range of 2.0-3.0 and anti-platelet therapy has been recommended.30 As combined therapy, VKA and anti-platelet therapy has not been shown to be superior to anti-platelet therapy alone, and since more major bleeding complications were observed in the combination group,33 we do not recommend up-front combined treatment with both VKA and anti-platelet therapy. Based on expert opinion and in slight contrast with international recommendations, we prefer treatment with either clopidogrel or VKA with an INR target range of 2.0-3.0 in these patients.33,34 In patients with a cerebral ischemic event (transient ischemic attack (TIA) or ischemic stroke) as clinical manifestation, there is no evidence supporting one therapy over the other. A prospective, comparative study in aPL-positive stroke patients showed no benefit of warfarin over aspirin (325 mg/day) on recurrent events; more (minor) haemorrhagic complications in the warfarin group were observed. However, these patients did not necessarily fulfil APS criteria.35 A small (n = 20), randomized, controlled trial in APS patients with ischemic stroke compared VKA and low-dose aspirin with low-dose aspirin alone, and demonstrated less recurrent stroke in the combined-therapy group.36 In APS patients with a first ischemic stroke or TIA without any cause other than APS on work-up, we propose treatment with either VKA with an INR target range of 2.0-3.0 or clopidogrel (since this is superior to aspirin for stroke prevention in a general population suffering from atherosclerotic cerebrovascular disease).37 Position of direct oral anticoagulants in APS DOACs such as dabigatran, rivaroxaban, apixaban, and edoxaban, were shown to be non-inferior to VKAs for treatment and secondary prevention of venous thromboembolic events and prevention of stroke and systemic embolism in patients with nonvalvular atrial fibrillation.38 Furthermore, these drugs have some advantages compared to VKAs as it is a fixed dose, no monitoring is required, interaction is limited, and there is a lower risk of intracranial and other major bleeding. Nonetheless, few studies are published concerning the use of DOACs in known APS. A recent review summarised the available data, including one randomized controlled trial (RAPS trial) and case series.39 From all available case series and case reports, 122 patients were analysed.39 High-risk APS patients with triple positivity or with several clinical criteria for definite APS, developed recurrent thrombosis more frequently while on DOACs in comparison to warfarin. The RAPS trial, comparing 54 APS patients treated with rivaroxaban to 56 APS patients treated with warfarin, demonstrated that the in vitro anticoagulant effect of rivaroxaban may be inferior to that of warfarin.40 A randomized controlled clinical trial in triple positive patients, comparing VKA treatment with rivaroxaban, was terminated early due to more thrombotic events in the rivaroxaban-arm, with particularly more ischemic strokes in the DOAC group.41 At this moment VKAs, compared to DOACs, remain the standard of care in the treatment of APS, especially in high-risk APS patients. DOACs might be an alternativetreatment modality for only those patients with instable INR or poor adherence to INR monitoring, and we anticipate more data from future trials using DOACs in thrombotic APS. Recurrent venous thrombotic events while using anticoagulation therapy Recurrent thrombosis in APS patients, despite adequate anticoagulation therapy (INR target range, 2.0-3.0) is common and occurs in up to one-third of all patients.2 However, recurrent thrombosis is uncommon in APS patients with a higher INR target range of 2.5-3.5 and most patients treated with a VKA with recurrent thrombosis, appear to have an INR target < 3.0.42 Therefore, anticoagulation therapy should be intensified with a INR target range of 2.5-3.5 in case of a recurrent venous event despite adequate INR. When a recurrent venous thrombosis is diagnosed with a suboptimal INR (< 2.0), therapeutic LMWH should be given for a period of two weeks and the INR treatment range should not be changed. Note that the INR value can be underestimated due to interference of thromboplastin and LAC, although this is mostly problematic with higher INR values, i.e. > 4.0.43 If the target INR cannot be reached and/or maintained with VKA, referral to an expert centre is recommended. Based on expert opinion, in patients with recurrent venous thrombosis, despite an adequate INR target range of 2.5-3.5 and after two weeks of therapeutic LMWH, additional long-term use of aspirin can be  recommended,  or  intensifying of  VKA  therapy with to an INR  target  range  of 3-4. Alternatively, a permanent switch to therapeutic LMWH can be considered. Combining VKA with hydroxychloroquine may also decrease the risk of recurrent venous thrombosis, although randomized studies with hydroxychloroquine in APS are still lacking.44,45 Until now, no clinical support for the use of statins in patients with recurrent venous thrombosis despite anticoagulation exists. However, based on in vitro work and surrogate endpoints, a beneficial role of statins has been suggested.46,47 Recurrent arterial thrombotic events while using anticoagulation therapy No consensus on treatment of recurrent arterial thrombosis was reached at the last meeting of the APS task force.30 Optimal treatment strategies for recurrent arterial thrombotic events have not been studied. In recurrent arterial thrombosis, including TIA or ischemic stroke, despite treatment with clopidogrel, we suggest switching treatment to VKA with an INR target range of 2.0-3.0. If recurrent arterial thrombosis occurs despite adequate treatment with VKA, referral to an expert centre is recommended. We do not recommend to routinely perform brain magnetic resonance imaging (MRI) in APS patients with an ischemic stroke. However, some experts believe that if neurological symptoms, including migraine or cognitive performance, deteriorate, a repeat brain MRI is indicated and new white matter abnormalities may lead to intensifying treatment. The possible benefits of this strategy have not been confirmed in clinical studies, and for patients with deterioration of neurological symptoms, referral to an expert centre is recommended.  Catastrophic antiphospholipid syndrome (CAPS) CAPS is a rare complication of APS and occurs in approximately 1% of APS patients. CAPS is a severe condition, including massive (mostly arterial) thrombosis of small vessels, causing multi-organ failure. Mortality in CAPS is high, up to 50%. Adequate and fast treatment initiation slightly improves clinical outcomes (mortality 20-40%) and treatment should be carried out in an expert centre.48,49 A combination of anticoagulant drugs (mostly unfractionated heparin with APTT ratio 2-2.5), intravenous corticosteroids (methylprednisolone 500-1000 mg/day for 3-5 days), therapeutic plasma exchange (TPE) and/or intravenous immunoglobulins (IVIG) (1 g/kg, for a time period of 3 days) is associated with highest survival rates. Combination of heparin-corticosteroids-plasmapheresis with or without additional IVIG results in 69-78% patient survival.50-52 TPE should be started when the clinical suspicion of CAPS arises, within a minimum of 5 days.52 Clinical response dictates the duration of TPE and no single clinical or laboratory parameter is used to determine when to discontinue treatment. For patients with CAPS and underlying SLE, treatment with cyclophosphamide (750 mg/m2 monthly) has been proposed.48,53 Rituximab (anti-CD20) has been administered to patients with refractory CAPS and may be of adjunctive value in a selected population of patients. Eculizumab (anti-complement 5) has been reported in case reports as a last resort, and could be considered in those patients refractory to all other therapies.54,55 Pregnancy and antiphospholipid antibodies Pregnancy complications of APS include recurrent first trimester pregnancy loss, intrauterine growth restriction (IUGR), preeclampsia (PET), premature birth, and intrauterine death (IUD). Early miscarriages are reported in 26-35% and aPL-related PET, premature birth or foetal death are seen in 10–20% of APS pregnancies.2,56 Women with positive aPL do not all carry the same obstetric risk. The following parameters are associated with an increased risk: the presence of LAC, more than one aPL (especially triple positivity), IgG aPL (instead of IgM), previous thrombosis, previous pregnancy complications, associated autoimmune condition, and hypocomplementemia.57 At present, however, risk stratification does not direct different treatment strategies. The current standard treatment, based on low-dose aspirin and LMWH, increases the percentage of a successful pregnancy from 20% to 54-80%.58 In patients with obstetrical APS, aspirin and a prophylactic dose of LMWH should be administered. Depending on the context (concurrent SLE, maternal age, non-criteria clinical features), treatment of patients with fewer than three miscarriages, and therefore, formally not classified as having APS, can be considered. To prevent overtreatment in these cases, confirmation of miscarriage by ultrasound is recommended. During pregnancy, regular clinical pregnancy follow-up is sufficient; only in specified subpopulations (e.g. patients with SLE, chronic kidney disease, morbid obesity, triple positive antibody profile, and patients with a thrombotic event during pregnancy), follow-up (of foetal growth and hypertensive pregnancy complications) should be intensified. Platelets should be checked at least once every 10-14 days after starting LMWH to exclude heparin-induced thrombocytopenia. If pregnancy complications (foetal death or miscarriage) still occur despite combined treatment with low-dose aspirin and prophylactic dose of LMWH, the dose of LMWH can be increased to a therapeutic dose; reverting back to a prophylactic dose of LMWH after 36 weeks of gestation can be considered. Based on expert opinion, patients with APS who already use therapeutic anticoagulation before pregnancy, should be switched to therapeutic dose of LMWH and low-dose aspirin should be added. Anti-factor Xa levels should be periodically monitored (at least once every trimester) in patients receiving therapeutic doses of LMWH, depending on the context. If this strategy fails, further treatment should take place in an expert centre. The recommendations are again based on expert opinion and retrospective cohort studies and include, for example, the addition of hydroxychloroquine or intravenous immunoglobulins. This should however, be confirmed in randomized controlled trials.59-61 In the postpartum period there is an increased risk for thrombosis. Patients with APS with an indication of therapeutic anticoagulation before pregnancy, should be switched to VKA or continue with therapeutic LMWH. Obstetrical APS patients should receive prophylactic or intermediate doses (a randomized clinical trial to investigate optimal dosing is currently underway62) of LMWH during a period of six weeks postpartum.57 Withdrawal of antithrombotic treatment in APS As mentioned above it is recommended to treat thrombotic APS patients lifelong. However, as long-term anticoagulation therapy is associated with haemorrhagic complications, the question arises whether it is possible to withdraw this therapy in selected patients, especially those who have a long-term event free period or who no longer have positive aPL, also called seroconversion. Only a few (uncontrolled and small) studies have addressed this question. Criado-Garcia et al described the effects of anticoagulation withdrawal in six primary APS patients in whom aPL had disappeared.63 None of these patients experienced a thrombotic event during a follow-up of 21 ± 4.9 months. It should be mentioned, however, that these patients were at low risk for recurrence, as they only had a venous thrombosis history. Similar results were found by Coloma Bazan et al, who reported no thrombotic recurrences after anticoagulation withdrawal durign a median follow-up of 20 months in 11 primary APS patients (seven with history of venous thrombosis and four with obstetrical APS).64 However, in a more recent study, 30 APS patients with anticoagulation withdrawal were compared to a thrombotic APS control population without withdrawal. During a median follow-up of 51 months, anticoagulation withdrawal was associated with a higher risk of thrombotic relapse (HR 4.82). Predictive factors were male gender, anti-β2GPI-positivity and triple positivity at onset as well as persistence positivity over time. Predictive factors for low risk of relapse were aspirin prescription and aPL disappearance during follow-up.65 In conclusion, data is insufficient to draw firm conclusions concerning anticoagulation withdrawal in APS patients. The available data suggests that anticoagulation could be withdrawn in APS patients with a single provoked venous thrombotic event in the presence of a known transient precipitating risk factor (such as smoking, disease activity, oral contraceptive use) together with disappearance of aPL. In all other situations, the high risk of thrombotic relapse favours the continuance of anticoagulation treatment. Randomized studies with larger sample sizes are still needed to confirm these statements.66,67 The decision to withdraw anticoagulation in APS patients should be made in an expert centre. Position of rituximab, hydroxychloroquine, intravenous immunoglobulins Several immunomodulatory drugs are suggested for the treatment for patients with APS, although their exact use in the treatment of APS is unclear; these include. rituximab, hydroxychloroquine (HCQ), and IVIG. Most evidence is based on cohort studies, case series or expert opinion. Only a few randomised controlled trials (RCTs) are published. Furthermore, their application is strongly dependent on the different clinical situations. For example, in secondary APS in SLE patients, HCQ is strongly recommended, as it has been shown that HCQ has ‘thrombo-protective’ effects, resulting in fewer venous and arterial thrombotic events.68 In primary APS, limited data is available. One retrospective cohort study demonstrated strong reduction of aPL titres and a decrease in the incidence of arterial thrombosis recurrence by using HCQ.69,70 One RCT randomizing 40 APS patients to VKAs versus VKAs with HCQ showed a protective effect of adding HCQ upon venous thrombotic events.45 At this moment, HCQ is not included in standard care of primary APS, but refractory cases, including refractory obstetrical APS, might benefit from this treatment. Treatment with HCQ (200-400 mg per day) is safe during pregnancy and lactation. Rituximab and IVIG may be used in difficult-totreat APS patients, especially in those with CAPS or recurrent haematological non-criteria manifestations as thrombocytopenia, but not as standard of care of APS.71,72 Prophylaxis in aPL-positive patients without earlier events The question remains whether patients with obstetric APS or individuals with positive aPL without thrombotic events should be treated to prevent thrombosis (primary thromboprophylaxis). At present, there is insufficient evidence to support prophylactic treatment for all of these patients.8,73 However, in patients with more risk factors for thrombosis (such as obesity, smoking, higher age) and/ or high-risk aPL profile (e.g., triple positive), low dose aspirin might be beneficial. In any case, attention should be paid to avoid or to treat any associated cardiovascular risk factors, e.g. using antihypertensives or cholesterollowering agents and avoidance of smoking, etc. Also, the administration of oral contraceptives should be used with caution and with counselling.74 Lastly, prophylaxis of venous thrombosis using LMWH is required for patients in situations associated with increased risk of thrombosis, such as surgical procedures, plaster casts, and those requiring bed rest.  CONCLUSION  APS is a rare and heterogeneous disease and as a result, well-designed and well-conducted clinical trials are scarce and the development of a formal guideline is difficult. However, by combining data from completed clinical intervention trials together with observational data and data from research in other thrombotic and/ or inflammatory conditions, recommendations for clinical practice can be formulated. Current national and international initiatives – such as the Dutch Arthritis Research and Collaboration Hub (ARCH) and the European Reference Network on Rare and Complex Connective Tissue and Musculoskeletal Diseases (ERN-ReConnet) – are aiming to structure the care for APS patients to offer a unique future opportunity to collect longitudinal clinical data on APS treatment and outcomes. Until a formal guideline has been made, this consensus paper fills the gap between evidence-based medicine and daily clinical practice for the care of APS patients. ACKNOWLEDGEMENTS KdL and ML performed the literature search and wrote the first article draft. All authors reviewed and commented on the first article draft and attended the first consensus meeting. JS and ML wrote the second article draft. All authors reviewed and commented on the second article draft. ML wrote the final report. All authors read the final report and agreed with the text. DISCLOSURES All authors declare no conflicts of interest. FUNDING This work was supported by the Arthritis Research and Collaboration Hub (ARCH) Foundation. REFERENCES Myakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Hemost. 2006;4:295-306. Cervera R, Piette JC, Font J et al. Antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of 1,000 patients. Arthritis Rheum. 2002;46:1019-27. Abreu MM, Danowski A, Wahl DG, et al. The relevance of "non-criteria" clinical manifestations of antiphospholipid syndrome: 14th International Congress on Antiphospholipid Antibodies Technical Task Force Report on Antiphospholipid Syndrome Clinical Features. Autoimmun Rev. 2015;14:401-14. Hughes GRV, Khamashta MA. Seronegative antiphospholipid syndrome. Ann Rheum Dis. 2003;62:1127. Galli M, Luciani D, Bertolini G, Barbui T. 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Research ArticleImmunology Free access | 10.1172/jci.insight.126471 Stem cell–derived tissue-associated regulatory T cells suppress the activity of pathogenic cells in autoimmune diabetes Mohammad Haque,1 Fengyang Lei,2 Xiaofang Xiong,1 Jugal Kishore Das,1 Xingcong Ren,3 Deyu Fang,4 Shahram Salek-Ardakani,5 Jin-Ming Yang,3 and Jianxun Song1 First published February 19, 2019 - More info Abstract The autoantigen-specific Tregs from pluripotent stem cells (PSCs), i.e., PSC-Tregs, have the ability to suppress autoimmunity. PSC-Tregs can be programmed to be tissue associated and to infiltrate into local inflamed tissues to suppress autoimmune responses after adoptive transfer. Nevertheless, the mechanisms by which the autoantigen-specific PSC-Tregs suppress the autoimmune response remain to be fully elucidated. In this study, we generated functional autoantigen-specific Tregs from the induced PSC (iPSCs), i.e., iPSC-Tregs, and investigated the underlying mechanisms of autoimmunity suppression by these Tregs in a type 1 diabetes (T1D) murine model. A double-Tg mouse model of T1D was established in F1 mice, in which the first generation of RIP-mOVA Tg mice that were crossed with OT-I T cell receptor (TCR) Tg mice was challenged with vaccinia viruses expressing OVA (VACV-OVA). We show that adoptive transfer of OVA-specific iPSC-Tregs greatly suppressed autoimmunity in the animal model and prevented the insulin-secreting pancreatic β cells from destruction. Further, we demonstrate that the adoptive transfer significantly reduced the expression of ICAM-1 in the diabetic pancreas and inhibited the migration of pathogenic CD8+ T cells and the production of the proinflammatory IFN-γ in the pancreas. These results indicate that the stem cell–derived tissue-associated Tregs can robustly accumulate in the diabetic pancreas, and, through downregulating the expression of ICAM-1 in the local inflamed tissues and inhibiting the production of proinflammatory cytokine IFN-γ, suppress the migration and activity of the pathogenic immune cells that cause T1D. Graphical Abstract Introduction Type 1 diabetes (T1D) develops due to autoimmune self-destruction of pancreatic β cells that produce insulin, and life-time administration of insulin is required for treatment of this disease (1). Although accumulating knowledge has contributed greatly to our understanding of the autoimmune pathogenesis of T1D, the precise causes remain unclear. It has been generally appreciated that autoimmune diseases arise from the breakdown of immune tolerance (2, 3). A number of studies found the massive infiltration of CD8+ T cells into the pancreases of newly diagnosed T1D patients whereas the number of CD4+ T cells was greatly reduced (4, 5). Infiltrated CD8+ T cells are pathogenic to the islets, can cause the destruction of pancreatic β cells, and ultimately reduce the secretion of insulin. Currently, there is no definitive treatment for controlling blood glucose level in T1D except durable insulin therapy. As a result, generation and transplantation of exogenous β cells to replace dead or dysfunctional endogenous β cells are considered as a promising strategy for controlling blood glucose level in patients with T1D. However, since the autoimmune disease is a continuous process, after β cell transplantation, diabetes may develop and progress again through destruction of the pancreatic islets by pathogenic T cells. Therefore, β cell transplantation might not be a long-lasting solution for the control of blood glucose level in T1D. Over the past several years, there has been an increasing interest in Tregs, which play a fundamental role in controlling various autoimmune responses. Numerous preclinical studies suggest that adoptive transfer of Tregs can prevent or cure T cell–mediated autoimmune diseases, such as T1D and arthritis (6, 7). There are several advantages of the Treg adoptive transfer over conventional treatments. These advantages include (a) the potential of antigen (Ag) specificity without general immunosuppression; (b) the option of inducing “physiological” long-lasting regulation in vivo; and (c) the possibility of Treg-based immunotherapy as a customized or personally designed agent for each patient, with reduced side effects (8, 9). It is now generally believed that adoptive transfer of in vitro–generated Tregs can reduce the hazards of complicated surgical events throughout the life. However, the use of Tregs has been complicated due to difficulties in expanding and characterizing this minor subset of T cells. Here, we report the development of a robust technique for producing a large amount of autoantigen-specific Tregs from induced pluripotent stem cells (iPSCs), i.e., iPSC-Tregs that retain all the quintessential characteristics of this T cell subset, including expressions of CD25, CTLA-4, and FoxP3 and production of IL-10. We show that adoptive transfer of these autoantigen-specific iPSC-Tregs significantly reduces the high ratio of CD8+ to CD4+ T cells in the pancreases of diabetic mice and markedly decreases the expression of intracellular adhesion molecule-1 (ICAM-1) in the pancreases of prediabetic and diabetic mice. These results demonstrate the great potential of stem cell–derived autoantigen-specific Tregs in T1D immunotherapy. Moreover, we show that the stem cell–derived tissue-associated Tregs control autoimmune diabetes via preventing the ICAM-1–mediated migration of pathogenic CD8+ T cells in the pancreas and suppressing the production of proinflammatory cytokine IFN-γ. Results Characterization of autoantigen-specific naturally occurring Treg–like iPSC-Tregs. Although FoxP3 is a master regulator and a specific molecular marker for naturally occurring Tregs (nTregs), there is evidence that FoxP3 expression is not a distinct and reliable marker or a sole regulator of functionally stable Tregs. In addition, recent evidence showed that various transcription factors are critical for the development of FoxP3+ Tregs, including Nr4a1, Ikzf4, Tnfrsf18, and Tbx21 (10). These genes, which are substantially expressed in nTregs, are essential for regulating Treg transcriptional programs and maintaining the lineage stability in Tregs. We previously showed the generation of functional Ag-specific iPSC-Tregs, which had the ability to suppress the development of autoimmune arthritis after adoptive transfer in murine models (11, 12). Because the iPSC-Tregs are retrovirally transduced with FoxP3 and TCR genes and have a similar phenotype to iPSC-Tregs in expression of IL-10 and TGF-β, we examined the Treg signature genes in the autoantigen-specific iPSC-Tregs. We adoptively transferred Rag 1–/– mice with pre-iPSC-Tregs that had been cocultured with OP9-DL1/DL4/I-Ab cells for 7 days. Six weeks later, mice were sacrificed and their spleens and lymph nodes (LNs) were removed for the isolation of CD4+CD25+ cells. Conversely, CD4+CD25+ cells were sorted from the spleens and LNs of normal C57BL/6 mice. RT-PCR analysis of the Treg signature genes showed that there were no significant differences between the mice in gene expression of the transcription factors (Nr4a1, Il2ra, Ikzf4, Tnfrsf18, and Bcl6) involved in the regulation of Treg functions (all P > 0.05, Figure 1). Collectively, these results and suggest that the autoantigen-specific iPSC-Tregs are nTreg-like suppressive cells. Figure 1 Characterization of autoantigen-specific nTreg-like iPSC-Tregs. PCR analysis was performed by TaqMan real-time PCR. Primers for sequences were used as follows: (A) Nr4a1, forward 5′-TGTGAGGGCTGCAAGGGCTTC-3′, reverse 5′-AAGCGGCAGAACTGGCAGCGG-3′; (B) Il2ra, forward 5′-AACTGCCAGTGCACCAGCAAC-3′, reverse 5′ GAGGTGGCTCCCTGCAGTGAC-3′; (C) Ikzf4, forward 5′-CAATCTGCTTCGCCACATCAAG-3′, reverse 5′-GCCACAGTAGTTGCACTTGTAG-3′; (D) Tbx21, forward 5′-GGAGCCCACTGGATGCGCCAG-3′, reverse 5′ AGGCAGCCTCTGGCTCTCCATC-3′, Tnfrsf18, forward 5′-CCTGCCAACCAGGCCAGAGGG-3′, reverse 5′-GTCCAAAGTCTGCAGTGACCG-3′; and (E) Bcl6, forward 5′-CACACCCGTCCATCATTGAA-3′, reverse 5′-TGTCCTCACGGTGCCTTTTT-3′. Data shown are representative of 3 identical experiments. The values represent mean ± SEM (n = 3). ns, P > 0.05, Student’s 1-tailed t test. Accumulation of activated CD8+ T cells in autoantigen-expressing pancreases of diabetic mice. To show the expression of autoantigen in the pancreases of double-Tg mice (B6-mOVA with OT-I), we performed immunofluorescence examination. The results showed the expression of OVA autoantigen in the double-Tg mice but not in the B6 or OT-I control mice (Figure 2A). Because the OVA-specific CD8+ T cells have the ability to migrate into the pancreases of the F1 generation that results from crossing B6 mOVA with OT-I TCR Tg mice and to cause destruction of the islets, we examined the fate of OVA-specific CD8+ T cells in vivo (Figure 2B). The migrations of CD8+ T cells in diabetic mice were further confirmed by flow cytometry in which more numbers of both CD8+ and TCRVβ5+ cells were present in diabetic mice (Figure 2C). In F1 mice after vaccinia virus (VACV) infection (Supplemental Figure 3; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.126471DS1), the draining LNs and spleens of the F1 mice were analyzed for the presence of OT-I CD8+ T cells. The proportions of OVA-specific CD8+ cells among the total CD8+ cells in the pancreatic LNs were significantly higher than in the pyloric, mesenteric, inguinal, cervical, or splenic LNs. No apparent accumulation or homing was observed in the non-Tg control B6 mice (Figure 2D). Additionally, all F1 mice after VACV infection developed autoimmune diabetes at age of 9 weeks, which was confirmed by the measurement of blood sugar (Figure 2E). These results verify that autoantigen-specific CD8+ T cells are the main pathogenic immune cells that induce autoimmune diabetes in the murine model. Figure 2 Accumulation of activated CD8+ T cells in autoantigen-expressing pancreases in diabetic mice. Pancreases were isolated from B6-mOVA × OT-I TCR double-Tg mice and B6 mice at 9 weeks of age, including 1 week in which mice were challenged with vaccinia viruses expressing OVA (VACV-OVA). (A) Detection of OVA expression by immunohistochemically staining. OVA expression (arrow) is indicated (original magnification, ×200). Data are representative of 5 mice per group in 3 independent experiments. Scale bar: 20.1 μm. (B) CD8+ T cell infiltration in the pancreas. CD8+ T cells (arrow) are indicated. Data are representative of 5 mice per group in 3 independent experiments. Scale bar: 20.1 μm. (C) OVA-specific CD8+ T cells in the pancreas. OVA-specific TCRVβ5 was analyzed by flow cytometry, after gating on CD8+ populations from the pancreas. Data shown are representative of 3 independent experiments (P < 0.001, Student’s 1-tailed t test). (D) Summarized analyses of OVA-specific CD8+ T cells in various locations. Data shown are representative of 5 mice per group in 3 independent experiments. Data shown are representative of 3 individual experiments. The values represent mean ± SD. **P < 0.01; ns, P > 0.05, multiple Student’s 1-tailed t test. (E) Blood glucose measurement. Data shown are representative of 3 individual experiments (n = 5). The values represent mean ± SD. **P < 0.01, Student’s 1-tailed t test. In vivo specificity of nTreg-like autoantigen-specific iPSC-Tregs. To exert their suppressive effects, Tregs need to migrate to specific tissues or organs, and this requires Ag specificity (8). We and others have previously reported that Tregs are detected within inflamed tissues and transplanted grafts, suggesting that these Tregs could control effector T cells in the peripheral tissues at sites of ongoing immune responses (12–14). Tregs may block the pathogenic cells from filling up their appropriate niche by taking up the space. The Treg population was reduced in autoimmune prone animals and patients, and Tregs were defective in NOD mice (15). In this study, we observed that mice receiving autoantigen OVA-specific iPSC-Tregs maintained a high population of both CD4+ and FoxP3+ cells in the pancreas, as compared with diabetic mice receiving control cells (iPSC-derived cells with empty vector) or nontissue-associated SM1-specific iPSC-Tregs by immunofluorescence examination (Figure 3A) and by flow cytometric analysis (90.1% vs. 12.5% or 16.8% at week 13, Figure 3B). These results suggest that the autoantigen-specific iPSC-Tregs are organ/tissue-associated nTreg-like suppressive cells. Figure 3 Accumulation of autoantigen-specific iPSC-Tregs in the pancreases of diabetic mice following adoptive transfer. B6-mOVA Tg × OT-I TCR double-Tg mice were immunized with VACV-OVA. At week 10, control cells or OVA- or SM1-specific pre-iPSC-Tregs were transferred into diabetic mice. Before or after the cell transfer, mice were sacrificed and their pancreases were isolated for analysis of CD4 and FoxP3. (A) Immunohistology at week 13 (original magnification, ×200). Scale bar: 20.1 μm. (B) Flow cytometric analysis at week 10 (before the cell transfer), 13, and 16. Data are representative of 5 mice per group in 3 independent experiments. Autoantigen-specific iPSC-Tregs can prevent destruction of pancreaticβcells and reduce autoimmune diabetes. Next, we injected diabetic mice with autoantigen-specific iPSC-Tregs. Two weeks after the cell transfer, blood glucose levels were measured. This cell transfer with autoantigen OVA-specific iPSC-Tregs but not control cells or nontissue-associated SM1-specific iPSC-Tregs significantly reduced blood glucose level with urine discharge in all of the diabetic mice (P < 0.0001, Figure 4A and Supplemental Figure 3). T1D develops due to the destructive autoreactive immune response in which CD8+ T cells play a critical role. CD8+ T cells infiltrated into the pancreatic islets of the T1D patients at both initial and final destructive phases of autoimmune β cells attack (16). The blood glucose level was increased because of the destruction of β cells in the islets by pathogenic CD8+ T cells in the diabetic pancreas, which was evidenced in the above mouse diabetic model. More CD8+ T cells were accumulated in the pancreases of mice receiving control cells or nontissue-associated Ag-specific iPSC-Tregs than autoantigen-specific iPSC-Tregs by immunofluorescence examination (Figure 4B) and by flow cytometric analysis (49.8% or 46.1% vs. 6.5%, Figure 4C). Only mice receiving autoantigen-specific iPSC-Tregs had a significantly reduced percentage of incidence of diabetes (P < 0.001, Figure 4D). A large number of inflammatory cells had infiltrated into the pancreases of mice receiving control cells, whereas the infiltration was substantially reduced in mice receiving autoantigen-specific iPSC-Tregs, as shown by H&E staining (Figure 4E). The total number of the islets was also reduced in mice receiving control cells as compared with autoantigen-specific iPSC-Tregs (P < 0.01 or P < 0.001, Figure 4F). To detect the destruction of β cells in the islets of diabetic mice, we stained pancreases with insulin. The islets were reduced in size and number in mice receiving control cells, whereas mice receiving autoantigen-specific iPSC-Tregs were protected from the destruction, by immunofluorescence examination (Figure 4G). The islets were partially destructed by pathogenic CD8+ T cells, which caused the increase of blood sugar and developed autoimmune diabetes, but some insulin+ staining in diabetic mice was still present. Collectively, these results indicate that autoantigen-specific iPSC-Tregs are specific and effective in protecting the hosts from islet destruction and in promoting insulin secretion to prevent the mice from diabetes mellitus. Figure 4 Prevention of destruction of pancreatic β cells and suppression of autoimmune diabetes by autoantigen-specific iPSCs-Tregs. Control cells or pre-iPSC-Tregs were adoptively transferred into diabetic mice, as described in Figure 3. (A) Blood sugar measurement at various weeks. Data shown are representative of 5 mice per group in 3 independent experiments. **P < 0.001; ***P < 0.0001, 2-way ANOVA analysis. (B) Immunofluorescence detection of pathogenic immune cells. Mice were sacrificed and pancreases were isolated for immunohistochemistry staining with CD8+ T cells (original magnification, ×200). Scale bar: 20.1 μm. Data are representative of 5 mice per group in 3 independent experiments. (C) Flow cytometric analysis of pathogenic immune cells. Pancreatic lymph nodes (LNs) were isolated, and single-cell suspensions were prepared for OVA-specific CD8+ TCRVβ5+ staining and analyzed by flow cytometry. Data shown are representative of 3 identical experiments (P < 0.001, Student’s 1-tailed t test). (D) Percentage of incidence of diabetes at week 22. Data shown are representative of 5 mice per group in 3 independent experiments. The values represent mean ± SD. **P < 0.001; ***P < 0.001, 1-way ANOVA analysis. (E) Representative photomicrographs (H&E staining) of the islet inflammation (original magnification, ×200). Scale bar: 20.4 μm. Cellular infiltrations (arrow) are indicated. Data are representative of 5 mice per group in 3 independent experiments. (F) Islet count from sections of 5 individual pancreases in each group. Data are represented as the mean ± SD of 3 independent experiments (***P < 0.001, 1-way ANOVA analysis). (G) Representative photomicrographs (immunofluorescence staining) of islet destruction (original magnification, ×200). Scale bar: 20.1 μm. Insulin-producing cells (arrow) are indicated. Data are representative of 5 mice per group in 3 independent experiments. Autoantigen-specific iPSC-Tregs can decrease the expression of ICAM-1. A number of mononuclear cells, including macrophages and CD4+ T cells, were found to be initially predominant in the autoimmune process taking place in T1D (17, 18). In autoimmune diabetes, these cells played an important role in the early infiltrating process and were shown to express ICAM-1, which served to bind to lymphocytes and possibly to monocytes and polymorphonuclear leukocytes (19). The infiltrated ICAM-1 can promote the release of several cytokines that modulate the expression of the adhesion molecules, thus increasing the adhesion of leukocytes and other autoreactive T cells (20). These autoreactive T cells, after migrating into the pancreas, start to destroy β cells. We thus examined the expression of ICAM-1 in the pancreases. The high expression of ICAM-1 in the pancreases of the diabetic mice, which was undetectable in the normal pancreases, was significantly reduced in diabetic mice receiving autoantigen-specific iPSC-Tregs but not other control cells (Figure 5, A and B). In addition, the high expression of ICAM-1 on the effector CD4+ T cells in the pancreases of the diabetic mice receiving control cells (84.4% or 78.9%), which was undetectable in the normal pancreases (0.35%), was markedly reduced in those of mice receiving autoantigen-specific iPSC-Tregs (18.4%) (Figure 5C). In addition, the expression of ICAM-1 on CD11b+ cells in the pancreases of diabetic mice receiving control cells (15.64% or 12.83%), which was at a low level in the normal pancreases (4.89%), was considerably reduced in those of mice receiving autoantigen-specific iPSC-Tregs (5.68%) (Figure 5D). Because CD11b is expressed on the surface of various leukocytes, including monocytes/macrophages, granulocytes, neutrophils, NK cells, and dendritic cells, the reduction of CD11b+ICAM-1+ cells indicated the inhibition of leukocyte adhesion and migration to mediate the inflammatory response. Taken together, these results suggest that the autoantigen-specific iPSC-Tregs can reduce the expression of ICAM-1 in diabetic mice, inhibit the migration of autoreactive CD8+ T cells into the pancreas, and, ultimately, protect the pancreatic β cells from destruction. Figure 5 Reduction of ICAM-1 expression in the diabetic pancreas by autoantigen-specific iPSC-Tregs. Mice were sacrificed 4 weeks after adoptive cell transfer, and the pancreases were isolated from nondiabetic, diabetic (cell transfer control), and diabetic (Ag-specific iPSC-Treg transfer) mice. Samples were prepared for immunohistochemistry staining. (A) ICAM-1+ zones (arrow) are indicated (original magnification, ×200). Data are representative of 5 mice per group in 3 independent experiments. (B) Quantification of ICAM-1 expression from sections of 5 individual pancreases in each group. Data are represented as the mean ± SD from 3 independent experiments (**P < 0.01; ***P < 0.001, 1-way ANOVA). (C and D) Flow cytometry analysis of ICAM-1 expression in pancreatic CD4+ T or CD11b+ cells. The pancreatic lymph nodes (LNs) were isolated, and single-cell suspensions were prepared for ICAM-1 staining on CD4+ or CD11b+ cells and analyzed by flow cytometry. Data shown are representative of 3 identical experiments. Autoantigen-specific iPSC-Tregs can migrate to the pancreas and produce a large amount of suppressive cytokines. In BDC2.5/NOD mice, FoxP3 was highly expressed in insulitic lesions where the function was impaired due to alterations in their gene expression profile (21, 22). We previously showed that the iPSC-Tregs exhibited a similar expression of Treg signature genes and produced IL-10 and TGF-β (12). In the current study, we observed very few numbers of FoxP3-expressing T cells in the pancreases of diabetic mice, and these cells did not secret IL-10 and TGF-β, which are the two key cytokines that are the hallmark for functional Tregs and secreted by activated and functional Tregs. We further showed that the pancreatic CD4+ T cells from mice receiving autoantigen-specific iPSC-Tregs produced substantially more IL-10 (66.1% vs. 12.9% or 15.1%) and TGF-β (5.61% vs. 0.62% or 1.68%), as compared with those from mice receiving control cells (Figure 6). These results support the assumption that autoantigen-specific iPSC-Tregs accumulating in the pancreas can secrete large amounts of suppressive cytokines (IL-10 and TGF-β), reduce the expression of ICAM-1, and prevent the migration of pathogenic CD8+ T cells into the pancreas, thus protecting the pancreas from destruction. Figure 6 Induction of IL-10 and TGF-β by autoantigen-specific iPSC-Tregs. Mice were sacrificed 4 weeks after adoptive cell transfer, and single-cell suspension was prepared from the pancreatic lymph nodes (LNs). Cells were stimulated with plate-coated CD3 and soluble CD28 antibodies and then stained with CD4, TGF-β, and IL-10. The CD4 population was gated, and the production of TGF-β and IL-10 was analyzed. Data shown are representative of 3 identical experiments. Autoantigen-specific iPSC-Tregs can decrease the production of IFN-γby pathogenic immune cells in the diabetic pancreatic islets. Treatment of NOD mice with an anti-IFN-γ monoclonal antibody could block diabetes (23); conversely, overproduction of IFN-γ in pancreatic islets provoked the disease (24). We next determined the secretion of IFN-γ in the diabetic islet. We found more IFN-γ–producing pancreatic CD8 and CD4 T cells in mice receiving control cells (31.4% and 30.4% or 29.7% and 28.6%); by contrast, both of the IFN-γ–producing pancreatic CD8 and CD4 T cells were significantly reduced in mice receiving autoantigen-specific iPSC-Tregs (14.6% and 10.9%) (Figure 7). These results indicate that the autoantigen-specific iPSC-Tregs can protect the islets from destruction by suppressing the secretion of IFN-γ produced by the pathogenic immune cells. Figure 7 Downregulation of IFN-γ by autoantigen-specific iPSC-Tregs in the diabetic pancreatic islets. Mice were sacrificed 4 weeks after adoptive cell transfer, and single cell suspension was prepared from the pancreatic lymph nodes (LNs). Cells were stimulated with plate-coated CD3 and soluble CD28 antibodies, and then stained with CD4, CD8, and IFN-γ for flow cytometric analysis. (A) IFN-γ production in CD4+ or CD8+ population. Data shown are the representative of 3 identical experiments. (B) Quantification of IFN-γ production. Data shown are representative of 3 identical experiments (**P < 0.01; ***P < 0.001, 1-way ANOVA). Discussion We and others have previously reported that adoptive transfer of stem cells has or stem cell–derived Tregs has the ability to suppress the development of autoimmunity in various animal models (11, 12, 25, 26). However, the suppressive mechanisms behind this suppression remain to be fully defined. By investigating the adoptive transfer of autoantigen-specific iPSC-Tregs in a murine model of T1D, we demonstrate that adoptive transfer significantly reduced the high ratio of CD8+ to CD4+ T cells in the pancreases of diabetic mice. We also show a critical role of this transfer in reducing the expression of ICAM-1 in inflamed pancreatic tissues and preventing the accumulation of pathogenic CD8+ T cells in the pancreases and diabetic pathogenesis. We further demonstrate that autoantigen-specific iPSC-Tregs associated with inflamed tissue and protected the islets from destruction through suppressing the production of proinflammatory cytokine IFN-γ. These findings may help us better understand the pathogenesis of autoimmunity in T1D and provide a foundation for therapeutic use of the stem cell–derived tissue-associated Tregs in the treatment of T1D (27). Among animal models of autoimmune diabetes, the NOD mouse is a widely used animal of autoimmune T cell–mediated T1D (spontaneously nonobese diabetic). The NOD mouse model clearly shows the leukocytic infiltration into the pancreatic islets (insulitis) and autoimmune destruction of the pancreatic β cell in female mice (2–4 weeks), which occurs later in male mice (5–7 weeks). Insulitis in NOD mice shows a combination of T cells (both CD4+ and CD8+), B cells (28), and inconstant numbers of macrophages/dendritic cells. In addition, a dominant-negative mutation in the mouse insulin 2 gene (Ins2Akita) produces a severe insulin deficiency syndrome exclusive of autoimmune participation and various transgenes overexpressed in β cells. Moreover, pharmacologically induced T1D (without autoimmunity) by alloxan or streptozotocin produces hyperglycemia in most strains of mice. Several low doses of streptozotocin combing direct β cell toxicity with local inflammation also elicit T1D in a male sex-specific fashion (29). To determine the suppressive mechanisms of stem cell–derived tissue-associated Tregs, we employed a mouse model of T1D by crossing B6-mOVA Tg mice with OT-I TCR Tg mice. Around 8 weeks of age, blood glucose levels were measured in F1 mice. Approximately 20%–40% of the mice were found to be diabetic; one potential explanation for this occurrence is that the OT-I CD8+ T cells may be tolerated during T cell development in the thymuses of F1 mice. As we propose in our model in which mice will develop autoimmune diabetes due to a large amount and the autoreactivity of CD8+ T cells, we activated tissue-associated CD8+ T cells by inoculating VACV-OVA into the mice in which the viruses-induced CD8+ T cells became highly polyfunctional (30). All mice developed diabetes with high blood glucose levels (Supplemental Figure 3) and more urine discharge, for which mice needed additional animal care. In this mouse model of T1D, OVA protein serves as inflamed tissue–associated autoantigen and is highly expressed in the pancreas. OVA-specific CD8+ T cells act as central pathogenic immune cells that accumulate in the pancreas and cause pancreatic β cell destruction and T1D (Figure 2). In addition, in this mouse model of T1D, we observed a non-OVA-specific CD4+ population existing in the pancreas (Figure 3), and these CD4+ T cells produced IFN-γ (Figure 7), which might also involve to the disease pathogenesis. These FoxP3–CD4+ T cells in the pancreas were TCRVα2–/Vβ5–, indicating that T cell activation was not through OVA-specific TCR. Of note, the number of these effector CD4+ T cells was dramatically reduced in mice receiving OVA-specific but not SM1-specific iPSC-Tregs (Supplemental Figure 4). These results also indicate that tissue-associated (OVA-specific) iPSC-Tregs are more effective than nontissue-associated (SM1-specific) iPSC-Tregs. Because of the challenge of VACV-OVA, these non-OVA-specific CD4+ T cells mainly originating from the RIP-mOVA descent may undergo bystander activation (31, 32). However, addition studies are needed on the pathways leading to bystander T cell activation under the condition. Tregs are an important component of self-tolerance, and numerous studies have demonstrated the effects of Tregs on natural and induced autoimmune diseases in various mouse models (33, 34). Targeting Tregs as a treatment for autoimmune disorders is an attractive approach, as there is an emerging consensus that many patients suffering from autoimmune disease have dysfunctional Tregs (35, 36). Given the manageability of murine models, the evidence for Tregs in control of autoimmune diseases has recently become clearer, particularly as a therapeutic intervention. In well-established models of rheumatoid arthritis (37), multiple sclerosis (38), and systemic lupus erythematosus (39), adoptive transfer of polyclonal Tregs could prevent or slow disease progression when administered prior to disease occurrence. In multiple sclerosis, Ag-specific Tregs targeting the disease-associated Ags were highly efficacious in reverting the ongoing disease (8, 40). Specifically, the nonobese T1D NOD.Cd28–/– mouse model deficient in Tregs developed diabetes at an accelerated rate as compared with NOD mice. Injection of NOD Tregs into NOD.Cd28–/– mice could delay and, in some cases, prevent the development of diabetes (41). In the current study, we have confirmed that adoptive transfer of stem cell–derived autoantigen-specific Tregs but not control cells, nonspecific iPSC-Tregs, or nontissue-associated Ag-specific iPSC-Tregs caused a high level of accumulation of these cells in the pancreas and reversed the disease process of autoimmune diabetes in the mouse model of T1D that was caused by pathogenic CD8+ T cells (Figure 4 and Supplemental Figure 3). A small number of Ag-specific iPSC-Tregs was observed in LNs and spleen (Supplemental Figure 5). A possible working mechanism of stem cell–derived autoantigen-specific Tregs is mediated through the secretion of large amounts of suppressive cytokines (e.g., IL-10 and TGF-β) that were identified (Figure 6). Of note, Ag-specific iPSC-Tregs have a functional stability for which we had previously demonstrated in an autoimmune condition (12), and FoxP3 expression in these cells persisted for 6 weeks after adoptive transfer in the diabetic mice (Figure 3). We further show that ICAM-1 (also known as CD54), a member of the Ig-like superfamily of adhesion proteins, plays a critical role in the accumulation of the pathogenic CD8+ T cells in the pancreases of the mice with autoimmune diabetes. ICAM-1 is a cell surface glycoprotein expressed in endothelial cells and some immune cells. ICAM-1 expressed in diabetic pancreatic tissues can bind to integrins of type CD11a/CD18 or CD11b/CD18 on pathogenic CD8+ T cells, and this binding facilitates the accumulation of pathogenic CD8+ T cells in the panaceas. The expression of ICAM-1 in the pancreases of diabetic mice is much higher than that in normal mice. In contrast, in the pancreases of diabetic mice, the adoptive transfer of autoantigen-specific iPSC-Tregs can dramatically reduce the expression of ICAM-1. This cell transfer can also reduce the accumulation of autoreactive CD8+ T cells in the pancreases and eventually protect the pancreatic β cells from further devastation (Figure 5). This conclusion is in line with those of previous studies showing that the expression of ICAM-1 is critical for the development of experimental autoimmune encephalomyelitis (42, 43). The role of IFN-γ, a prototypical Th1 cytokine, has been appreciated in the development of T1D, because the treatment of NOD mice with an anti-IFN-γ monoclonal antibody prevented the development of the autoimmune diabetes (23, 24). We detected an elevated amount of IFN-γ in both effector CD8 and CD4 T cells of the diabetic islets and showed that adoptive transfer of stem cell–derived autoantigen-specific Tregs markedly decreased the production of the proinflammatory cytokine in the pathogenic T cells. We demonstrated that the autoantigen-specific iPSC-Tregs protected the islets from mass destruction via suppressing the production of pathogenic IFN-γ (Figure 7). As other proinflammatory cytokines, such as IL-21 (44), IL-17 (45), TNF-α (46), and IL-1β (47) also play roles in the pathogenesis of T1D, studies on these proinflammatory cytokines would be needed to better exploit the stem cell–derived tissue-associated Tregs as a therapeutic approach against autoimmune disorders. The Tg model used in this study highly expressed OVA as surrogate autoantigen and received approximately 2 million of OVA-specific iPSC-Tregs for the treatment, which may be much higher than in the clinical setting with diverse low-abundance target peptides. However, the understanding of islet Ags has previously resulted in translation into new strategies by targeting tissue-specific immune interventions to prevent disease progression as well as to reverse T1D. A big advantage of our approach is the development of large numbers of autoantigen-specific nTreg-like suppressive cells (Figure 1 and Supplemental Figures 1 and 2), which can accumulate in the inflamed tissues and suppress local pathogenic immune cells. In addition, the stem cell–derived Tregs maintain stability for up to 3 months (11, 12), because the overexpression of FoxP3 has the ability to suppress the potential switch of Tregs to Th17 cells. Of note, the selection of optimal autoantigens for the development of tissue-associated Tregs is critical for the success of the Treg-based T1D immunotherapy. Taken together, the current study provides insight into the mechanisms by which stem cell–derived tissue-associated Tregs suppress autoimmunity in T1D and suggests that stem cell–derived autoantigen-specific Tregs have a great potential to be adapted as an immunotherapy for T1D. Methods Cell lines and mice. The mouse iPS-MEF-Ng-20D-17 cell line was obtained from the RIKEN Cell Bank (48). The OP9-DL1/DL4/I-Ab cell line was generated by a retroviral transduction of the OP9 cells (12). The SNL76/7 cell line (ATCC SCRC-1049) was purchased from ATCC. C57BL/6 (B6), Rag1–/–, B6 mOVA RIP (RIP-mOVA) Tg, and OT-I TCR Tg mice were purchased from The Jackson Laboratory. Cell culture. iPSCs were maintained on feeder layers of irradiated SNL76/7 cells in 6-well culture plates (Nunc) and were passaged every 3 days (48). Retroviral transduction and generation of OVA-specific iPSC-Tregs. cDNA for FoxP3 with OVA 322–339 (ISQAVHAAHAEINEAGR)-specific I-Ab-restricted TCR genes (Vα2 and Vβ5; obtained from Dario A. Vignali, University of Pittsburgh, Pittsburgh, Pennsylvania, USA) or LCMV (SMARTA1; SM1) gp61 (GLKGPDIYKGVYQFKSVEFD)-specific I-Ab-restricted TCR genes (Vα2 and Vβ8; obtained from Matthew A. Williams, University of Utah, Salt Lake City, Utah, USA) was used for retroviral transduction of mouse iPSCs and the generation of OVA- or SM1-specific iPSC-Tregs (12). Antibodies. PE-, PE/Cy7-, Alexa Fluor 647–, APC-, or APC/Cy7-conjugated anti-mouse TCRVβ5 (MR9-4), CD4 (GK1.5), CD11b (M1/70), TGF-β1 (TW7-16B4), and FoxP3 (MF-14) were obtained from Biolegend. FITC- or PE-conjugated anti-mouse CD8 (6A242) was obtained from Santa Cruz Biotechnology. APC-conjugated IL-10 (JES5-16E3) was purchased from BD Biosciences. Rabbit insulin antibody (C27C9; 3014) was purchased from Cell Signaling and anti-ICAM1 (YN1/1.7.4; ab25375) antibody was obtained from Abcam. Flow cytometric analysis. T cells from the pancreatic LNs were collected and intracellular IL-10 and TGF-β were analyzed by flow cytometry after gating on live CD4+ or CD8+ cells. RT-PCR. MiDR OTII 2A FoxP3-transduced mouse iPSCs were cocultured with OP9-DL1-DL4-I-Ab in the presence of mFlt-3L for 7 days. Differentiated iPSCs were separated and adoptively transferred into Rag1–/– mice. Mice were housed for 6 weeks for in vivo maturation of the iPSC-Tregs. Six weeks later, spleens and LNs were collected from normal B6 mice and cell transferred Rag1–/– mice. CD4+CD25+ cells were sorted and total RNA was extracted from sorted cells using QIAgen RNeasy mini kits. Samples were subjected to reverse transcription using a high-capacity cDNA synthesis kit (Applied Biosystems). PCR analysis was performed by TaqMan real-time PCR (Thermo Fisher Scientific). Ex vivo stimulation assay. Mice were sacrificed 4 weeks after adoptive cell transfer and single-cell suspension was then prepared from the pancreatic LNs. Cells were stimulated with plate-coated CD3 plus soluble CD28 antibodies. Productions of IL-10 and TGF-β were determined by intracellular cytokine staining (12). Murine autoimmune diabetes model. Autoimmune diabetes was induced in F1 mice that were crossed between RIP-mOVA Tg with OT-I TCR Tg mice by i.p. injection with VACV-OVA (2 × 106 PFU/mouse) (49). All of the mice developed diabetes after viral injection. Blood glucose was measured 1 week after the VACV-OVA injection. Blood glucose measurement. Blood glucose levels were determined using a Glucometer Ascensia Elite XL (Bayer). Six hundred milligrams per deciliter is the maximum measurable glucose reading. Mice were typically considered diabetic with readings of >250 mg/dl. Adoptive cell transfer. B6-mOVA Tg × OT-I TCR double-Tg mice (F1) were immunized with VACV-OVA for 1 week. At week 10, OVA-specific pre-iPSC-Tregs or iPSC-derived control cells (3 × 106) were i.v. transferred into diabetic mice. Four to six weeks later, mice were euthanized, and the pancreatic tissues were removed for histopathological examination. Histology and immunohistochemistry. For H&E staining, pancreatic tissues were fixed with 10% neutral formalin solution (VWR), and the fixed samples were prepared and stained as described previously (48). For immunofluorescent microscopy, the pancreatic tissues were frozen in cryovials on dry ice immediately after resection. Cryosectioning and immunofluorescent staining were performed as described previously (48). Statistics. Multiple Student’s 1-tailed t test or 1-way ANOVA or 2-way ANOVA analysis was performed to analyze the differences between the groups, using GraphPad Prism. A P value of less than 0.05 was considered significant. Study approval. The present studies in mice were reviewed and approved by The Texas A&M University Animal Care Committee and were in accordance with the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care. Author contributions JS and JMY designed the experiments, analyzed data, and contributed to the writing of the paper. MH, FL, and JKD performed the experiments. DF and SSA provided reagents in the animal model. XX and XR analyzed data. Supplemental material View Supplemental data Acknowledgments JS is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. This work was supported by National Institutes of Health grants R01AI121180, R01CA221867, and R21AI109239 and the American Diabetes Association (1-16-IBS-281 to JS). Footnotes Conflict of interest: The authors have declared that no conflict of interest exists. Copyright: © 2019 American Society for Clinical Investigation Reference information: JCI Insight. 2019;4(7):e126471. https://doi.org/10.1172/jci.insight.126471. References Delovitch TL, Singh B. The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity. 1997;7(6):727–738. 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Journal of Immunology Research is a peer-reviewed, Open Access journal that provides a platform for scientists and clinicians working in different areas of immunology and therapy. The journal publishes research articles, review articles, as well as clinical studies related to classical immunology,...

Objective We examined genome-wide DNA methylation changes in CD8+ T cells from patients with lupus and controls and investigated the functional relevance of some of these changes in lupus. Methods Genome-wide DNA methylation of lupus and age, sex and ethnicity-matched control CD8+ T cells was...

Drug-induced lupus (DIL) is a side effect of long-term treatment with various medications, an idiosyncratic adverse drug reaction (ADR) that has been documented for some seven decades. While symptoms can be severe, DIL, unlike systemic lupus erythematosus (SLE), is fully reversible simply by...