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Variants in STAT4 are associated with systemic lupus erythematosus (SLE) and other autoimmune diseases. How disease-associated variants affect STAT4 expression, in particular in CD4 T cells where STAT4 plays an essential role, is unknown.We compared Th1 differentiation between naïve CD4 T cells from healthy donors (HD) homozygous for the risk (R/R) or non-risk (NR/NR) alleles. We analyzed epigenetic marks in STAT4 and evaluated the relevance of its third intron. We assessed the consequences of Stat4 overexpression in vivo. We analyzed the effects of the STAT4 genotype in patients with lupus nephritis.Naïve CD4 T cells from NR/NR HD, downregulated STAT4 in response to IL-12. In contrast, cells from R/R HD maintained high levels. R/R cells exhibited higher abundance of transcriptionally active STAT4 and increased IFN-γ production. Accordingly, R/R HD exhibited a stronger induction of local active enhancer marks. Genetic editing confirmed the presence of a negative regulatory region in the STAT4 third intron, where most of the SLE-associated STAT4 SNPs are located. In vivo forced expression demonstrated that increases in Stat4 levels in T cells enhanced glomerulonephritis. Accordingly, the R/R genotype was associated with suboptimal response to treatment and with worse clinical outcomes in patients with proliferative lupus nephritis.The SLE-associated STAT4 haplotype is associated with abnormal IL-12-mediated STAT4 transcriptional regulation. Carriers of the risk variant exhibit exaggerated CD4 pro-inflammatory capacities that, in the context of SLE, contribute to a more severe disease. R/R patients may benefit from blockade of the IL-12-STAT4 pathway. This article is protected by copyright. All rights reserved.

The cellular origin of CD4− CD8− (double negative, DNT) TCR-α/β+ T cells remains unknown. Available evidence indicates that they may derive from CD8+ T cells, but most published data have been obtained using cells that bear an invariant transgenic T cell receptor that recognizes an Ag that is not present in normal mice. Here, we have used complementary fate mapping and adoptive transfer experiments to identify the cellular lineage of origin of DNT cells in wild-type mice with a polyclonal T cell repertoire. We show that TCR-α/β+ DNT cells can be traced back to CD8+ and CD4+CD8+ double positive cells in the thymus. We also demonstrate that polyclonal DNT cells generated in secondary lymphoid organs proliferate upon adoptive transfer and can regain CD8 expression in lymphopenic environment. These results demonstrate the cellular origin of DNT cells and provide a conceptual framework to understand their presence in pathological circumstances.

COVID-19 can range from asymptomatic to life-threatening. Early identification of patients who will develop severe disease is crucial. A number of scores and indexes have been developed to predict severity. However, most rely on measurements not readily available. We evaluated hematological and biochemical markers taken on admission and determined how predictive they were of development of critical illness or death. We observed that higher values of readily available tests, including neutrophil:lymphocyte ratio; derived neutrophil index; and troponin I were associated with a higher risk of death or critical care admission (P

Activation of self-reactive CD8+ T cells induces a peripheral tolerance mechanism that involves loss of CD8 expression. Because genetic deficiency of Fas and Fasl causes the accumulation of double-negative (DN; CD3+ TCR-αβ+ CD4- CD8-) T cells that have been proposed to derive from CD8+ cells, we decided to explore the role of Fas and FasL in self-antigen-induced CD8 downregulation. To this end, we quantified Fas and FasL induction by different stimuli and analyzed the effects of Fas/FasL deficiency during a protective immune response and after exposure to self-antigens. Our data describes how Fas and FasL upregulation differs depending on the setting of CD8 T cell activation and demonstrates that Fas/FasL signaling maintains CD8 expression during repetitive antigen stimulation and following self-antigen encounter. Together, our results reveal an unexpected role of Fas/FasL signaling and offer a new insight into the role of these molecules in the regulation of immune tolerance.

CD8 T cells can kill malignant cells in an antigen-specific manner. However, anti-tumoral responses are usually limited by suppressive factors that curb the effector responses of tumor-infiltrating CD8 T cells. Therapeutic strategies to overcome intra-tumoral T cell suppression, for example immune checkpoint inhibition, have been clinically effective in patients with cancer. Here, we provide data that demonstrates that GK-1, a peptide derived from the parasite Taenia crassiceps, promotes an anti-melanoma CD8 T cell response with heightened effector characteristics that leads to an increased amount of tumor-infiltrating CD44+ IFN-γ-producing CD8 T cells. The response induced by GK-1 was associated with a reduction in the expression of PD-1 and PD-L1 on tumor-infiltrating CD8 and dendritic cells, respectively, effects that led to a dramatic decrease in tumor burden. Our results suggest that the immunomodulatory properties of GK-1 may promote a CD8 T cell response that may be therapeutically useful in the setting of cancer.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease linked to profound defects in the function and phenotype of T lymphocytes. Here, we describe abnormal signaling pathways that have been documented in T cells from patients with SLE and discuss how they impact gene expression and immune function, in order to understand how they contribute to disease development and progression.

TCR-αβ(+) double negative (DN) T cells (CD3(+) TCR-αβ(+) CD4(-) CD8(-) NK1.1(-) CD49b(-) ) represent a minor heterogeneous population in healthy humans and mice. These cells have been ascribed pro-inflammatory and regulatory capacities and are known to expand during the course of several autoimmune diseases. Importantly, previous studies have shown that self-reactive CD8(+) T cells become DN after activation by self-antigens, suggesting that self-reactive T cells may exist within the DN T-cell population. Here, we demonstrate that programmed cell death 1 (PD-1) expression in unmanipulated mice identifies a subset of DN T cells with expression of activation-associated markers and a phenotype that strongly suggests they are derived from self-reactive CD8(+) cells. We also found that, within DN T cells, the PD-1(+) subset generates the majority of pro-inflammatory cytokines. Finally, using a TCR-activation reporter mouse (Nur77-GFP), we confirmed that in the steady-state PD-1(+) DN T cells engage endogenous antigens in healthy mice. In conclusion, we provide evidence that indicates that the PD-1(+) fraction of DN T cells represents self-reactive cells.

TCR-αβ+ double-negative (DN; CD4−CD8−) T cells represent a poorly understood cellular subset suggested to contribute to the pathogenesis of the autoimmune disease systemic lupus erythematosus. DN T cells have been proposed to derive from CD8+ cells. However, the conditions that govern the loss of CD8 expression after Ag encounter are unknown. In this study, we tracked the fate of CD8 T cells from transgenic TCR mice exposed to their cognate Ags as self or in the context of infection. We demonstrate that CD8 T cells lose CD8 expression and become DN only when cognate Ag is sensed as self. This process is restricted to tissues where the Ag is present. We also show that DN T cells derived from self-reactive CD8 cells express the inhibitory molecules PD-1 and Helios. These molecules identify a subset of DN T cells in normal mice. A similar population expands when CD8 T cells from repertoires enriched in self-reactive cells (Aire-deficient) are transferred into cognate hosts. Collectively, our data suggest that a subset of DN T cells, identified by the expression of PD-1 and Helios, represent self-reactive cells. Our results provide an explanation for the origin of DN T cells and introduce CD8 loss as a process associated with self-Ag encounter.

Background: In human immunodeficiency virus (HIV)-infection, persistent T-cell activa-tion leads to rapid turnover and increased cell death, leading to immune exhaustion and increased susceptibility to opportunistic infections. Stimulation of the vagus nerve in-creases acetylcholine (ACh) release and modulates inflammation in chronic inflammato-ry conditions, a neural mechanism known as the cholinergic anti-inflammatory pathway (CAP). Pyridostigmine, an ACh-esterase inhibitor, increases the half-life of endogenous ACh, therefore mimicking the CAP. We have previously observed that pyridostigmine re-duces ex-vivo activation and proliferation of T-cells obtained from people living with HIV. Methods: We conducted a 16-week proof-of-concept open trial using pyridostigmine as add-on therapy in seven HIV-infected patients with discordant immune response receiv-ing combined antiretroviral therapy (cART), to determine whether pyridostigmine would promote an increase in total CD4+ T-cells. The trial was approved by the Institutional Re-search and Ethics Board and registered in ClinicalTrials.gov (NCT00518154). Results: Seven patients were enrolled after signing informed consent forms. We ob-served that addition of pyridostigmine induced a significant increase in total CD4+ T-cells (baseline=153.1±43.1 vs. week-12=211.9±61.1 cells/µL; p=0.02). Post-hoc analysis showed that in response to pyridostigmine, four patients (57%) significantly increased CD4+ T-cell counts (responders=257.8±26.6 vs. non-responders=150.6±18.0 cells/µL; p=0.002), and the effect persisted for at least one year after discontinuation of pyri-dostigmine. Conclusion: Our data indicates that in patients with HIV, add-on pyridostigmine results in a significant and persistent increase in circulating CD4+ T-cells.

Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) is a costimulatory receptor in the immunoglobulin superfamily closely related to CD28 and ICOS, and binds to CD80 and CD86 (1;2). Expression of CTLA-4 is constitutive on regulatory T cells (3) and induced following activation on effector T cells (1). It exerts an essential inhibitory role and its absence causes an early and lethal autoimmune disease in mice (4). The CTLA4 gene is highly conserved (76% homology between human and mouse) (5). It is comprised of 4 exons: exon 1 codes for the signal peptide; exon 2 for the ligand-binding domain; exon 3 for the transmembrane region; exon 4 for the intra-cytoplasmic tail (Figure 1A) (6). Human peripheral blood lymphocytes express 3 isoforms of CTLA-4 produced by alternative splicing: full length CTLA-4 (flCTLA-4; all four exons), soluble CTLA-4 (exons 1, 2, and 4), and a short variant that lacks both the ligand-binding domain and the transmembrane domain (1/4 CTLA-4) (6). Allelic variations and single nucleotide polymorphisms in the CTLA4 gene have been associated to several human autoimmune diseases including autoimmune thyroid disease (6), rheumatoid arthritis (7) and systemic lupus erythematosus (SLE) (8;9). Interestingly, the polymorphisms associated with autoimmune disease affect CTLA4 splicing and thus the relative expression of each variant isoform (6). How the differential expression of CTLA-4 isoforms impacts susceptibility to autoimmune disease is not yet clear. Figure 1 The 1/4 CTLA-4 splice variant codes for a secreted protein The 1/4 CTLA-4 isoform lacks the CD80/86-binding domain and the transmembrane portion and thus its function remains unclear. Forced expression of the 1/4 CTLA-4 isoform in T cells was shown to induce spontaneous autoimmune disease and facilitate the development of experimental allergic encephalomyelitis in C57BL/6 mice through an unknown mechanism (10). Here we demonstrate that in lupus-prone mice increased expression of 1/4 CTLA-4 accelerates autoimmunity, exacerbates disease pathology, and causes early mortality.

Objective Systemic lupus erythematosus (SLE) is a chronic inflammatory disease associated with aberrant immune cell function. Treatment involves the use of indiscriminate immunosuppression, which results in significant side effects. SLE T cells express high levels of calcium/calmodulin-dependent protein kinase type IV (CaMKIV), which translocates to the nucleus upon engagement of the T cell receptor–CD3 complex and accounts for abnormal T cell function. The purpose of this study was to determine whether inhibition of CaMKIV would improve disease pathology. Methods We treated MRL/lpr mice with KN-93, a CaMKIV inhibitor, starting at week 8 or week 12 of age and continuing through week 16 and evaluated skin lesions, proteinuria, kidney histopathology, proinflammatory cytokine production, and costimulatory molecule expression. We also determined the effect of silencing of CAMK4 on interferon-γ (IFNγ) expression by human SLE T cells. Results CaMKIV inhibition in MRL/lpr mice resulted in significant suppression of nephritis and skin disease, decreased expression of the costimulatory molecules CD86 and CD80 on B cells, and suppression of IFNγ and tumor necrosis factor α production. In human SLE T cells, silencing of CAMK4 resulted in suppression of IFNγ production. Conclusion We conclude that suppression of CaMKIV mitigates disease development in lupus-prone mice by suppressing cytokine production and costimulatory molecule expression. Specific silencing of CAMK4 in human T cells results in similar suppression of IFNγ production. Our data justify the development of small-molecule CaMKIV inhibitors for the treatment of patients with SLE.

Objective To quantify the expression of CD44 and variant isoforms CD44v3 and CD44v6 on T cells from patients with systemic lupus erythematosus (SLE), and to assess correlations of the level of expression of these molecules with disease manifestations. Methods Information on clinical and demographic characteristics was collected, and blood samples were obtained from 72 patients with SLE and 32 healthy control subjects matched to the patients by sex, race, and age. Expression of CD44 and variants CD44v3 and v6 on T cell subsets was determined by flow cytometry, and Pearson's correlations of their expression levels with clinical variables, SLE Disease Activity Index (SLEDAI) scores, and presence of lupus nephritis were determined. Wilcoxon's rank sum tests and conditional multivariable regression analyses were applied to identify differences in the expression of CD44 between patients with SLE and healthy controls. Results Expression of CD44 was higher on CD4+ and CD8+ T cells from SLE patients compared with controls (P ≤ 0.03). Expression of CD44v3 and CD44v6 was also higher on total T cells and CD4+ and CD8+ T cells from SLE patients compared with controls (P ≤ 0.03). Cell surface levels of CD44v3 on total T cells, CD4+ T cells, and CD8+ T cells as well as cell surface expression of CD44v6 on total T cells and CD4+ T cells were correlated with the SLEDAI score (P < 0.05). The presence of lupus nephritis was associated with the expression of CD44v6 on total T cells, CD4+ T cells, and CD4−CD8− T cells (P < 0.05). Positivity for anti–double-stranded DNA antibodies was associated with the expression levels of CD44v6 on T cells (P < 0.05). Conclusion These results indicate that expression levels of CD44v3 and CD44v6 on T cells may represent useful biomarkers of SLE activity.

IL-2 plays a key role in setting the balance between immunity and tolerance. This cytokine has a dual role as the regulator of the two main phases of the immune response (proliferative and suppressive). Likewise, activation induced cell death and the induction and maintenance of regulatory T cells are the tolerance mechanisms regulated by IL-2, which convey the link between IL-2 abnormalities and the development of autoimmune disorders, such as systemic lupus erythematosus (SLE). Particularly, in SLE murine models and in humans, deficiency in IL-2 synthesis and activity has been proven. Diverse signaling pathways abnormalities (TCR, NF-kappaBeta, NF-AT) have been involved in the IL-2 transcriptional dysregulation displayed by T cells from SLE patients, and its functional relevance as part of the physiopathogenic scheme has been shown. Aberrant expression and activity of multiple IL-2 transcriptional factors, such as c-fos, and predominantly, CREM and CREB have been involved in this immune dysregulation. Diverse alterations in signaling kinases and phosphatases (PKA, PP2A, CAMKIV) and the modulation by epigenetic mechanisms have been related to the altered CREM/pCREB index. The synergic effect of multiple abnormalities in the transcriptional factors previously mentioned has been shown to be of functional relevance in lupus.

Multiple elements are known to participate in ischemia/reperfusion (I/R)-mediated tissue injury. Amongst them, B cells have been shown to contribute by the production of antibodies that bind to ischemic cells and fix complement. It is currently unknown whether B cells participate through antibody-independent mechanisms in the pathogenesis of I/R. In a mesenteric I/R model we found that B cells infiltrate the injured intestine of normal and autoimmune mice 2 h after reperfusion is established. B cell depletion protected mice from the development of I/R-mediated intestinal damage. The protection conferred by B cell depletion was significantly greater in MRL/lpr mice. Finally, we show that ischemic tissue expressed the B cell-attractant CXCL13 and infiltrating B cells expressed the corresponding receptor CXCR5. Our data grant B cells an antibody-independent role in the pathogenesis of intestinal I/R and suggest that B cells accumulate in the injured tissue in response to the chemokine CXCL13.

SummaryThe emerging role of interleukin (IL)-17 as a hallmark proinflammatory cytokine of the adaptive immune system, produced primarily by a new T helper cell subset termed ‘Th17’, has received considerable attention. Differentiation of Th17 cells is driven by the simultaneous presence of transforming growth factor-β and certain inflammatory cytokines (e.g. IL-6, IL-21), and recent studies have shown that inflammation instigated by IL-17-producing cells is central to the development and pathogenesis of several human autoimmune diseases and animal models of autoimmunity. In this review, we focus on the information regarding IL-17 and systemic lupus erythematosus (SLE), a chronic autoimmune disease. The work that has explored the development and behaviour of IL-17-producing cells in SLE is discussed, and different mechanisms by which IL-17 could potentially augment inflammation and autoantibody production in the context of SLE are proposed.

Double negative (DN) T cells are expanded in patients with systemic lupus erythematosus (SLE) and stimulate autoantibody production as efficiently as CD4+ T cells. In this study, we demonstrate that DN T cells from patients with SLE produce significant amounts of IL-17 and IFN-γ, and expand when stimulated in vitro with an anti-CD3 Ab in the presence of accessory cells. Furthermore, IL-17+ and DN T cells are found in kidney biopsies of patients with lupus nephritis. Our findings establish that DN T cells produce the inflammatory cytokines IL-17 and IFN-γ, and suggest that they contribute to the pathogenesis of kidney damage in patients with SLE.

T cells from patients with systemic lupus erythematosus (SLE) display a number of biochemical abnormalities which include altered expression of key signaling molecules, heightened calcium responses, and skewed expression of transcription factors. These defects are involved in the altered behavior of SLE T cells and are probably central in the disease pathogenesis. The aim of this communication is to review the defects that have been consistently documented in SLE T cells, highlighting molecules and pathways that represent therapeutic targets.

Background Transplant recipients develop antibodies against a wide array of HLA specificities, and not only against the antigens to which they have been exposed. The aim of the present study was to establish if HLA-unrelated immune stimulation is capable of triggering the production of HLA antibodies. Methods We determined the presence of HLA antibodies in 20 healthy adults before and after the administration of an immune stimulus (hepatitis B vaccine plus tuberculin skin test). HLA antibodies were assessed with a flow-cytometry based system. Results At baseline, HBsAg antibodies were detected in protective titers in 75% of the subjects; HLA antibodies were negative in all but one. One week after the antigenic stimulus, HBsAg antibody titers increased significantly, without any detectable changes in HLA antibodies. Surprisingly, HLA antibodies developed in 8 participants one month after the application of the stimulus. One additional subject developed HLA antibodies one month later. Therefore 9/20 subjects became PRA positive during the observation period. Antibody specificities were identified by single antigen assay. The alloantibody response elicited by the immune stimulus was not consistent with memory cell stimulation. Conclusions The findings of this study demonstrate that a non-HLA antigenic stimulus is capable of eliciting the development of HLA antibodies in healthy adults.

Homeostasis of the immune system depends on the proper function of regulatory T cells (T(reg) cells). Compromised suppressive activity of T(reg) cells leads to autoimmune disease and graft rejection and promotes anti-tumor immunity. Here we report a previously unrecognized requirement for the serine-threonine phosphatase PP2A in the function of T(reg) cells. T(reg) cells exhibited high PP2A activity, and T(reg) cell-specific ablation of the PP2A complex resulted in a severe, multi-organ, lymphoproliferative autoimmune disorder. Mass spectrometry revealed that PP2A associated with components of the mTOR metabolic-checkpoint kinase pathway and suppressed the activity of the mTORC1 complex. In the absence of PP2A, T(reg) cells altered their metabolic and cytokine profile and were unable to suppress effector immune responses. Therefore, PP2A is required for the function of T(reg) cells and the prevention of autoimmunity.

The immunological hallmark of SLE is B cell hyperactivity. CD154 (CD40-L) is normally expressed in activated T cells, and plays an important role in T-B interactions. Its expression is increased in SLE T cells. Additionally, its expression on B cells leads to the development of SLE-like disease in a transgenic model. IL-10 is a key cytokine in the disturbed SLE immune system. The aim of this work was to explore the relation between IL-10 and CD154 expression in SLE B cells. We studied 11 SLE patients and 10 healthy volunteers. Mononuclear cells were isolated from peripheral blood and cultured in the presence or absence of Cowan I Strain Staphylococcus (CSS). Surface CD154 and intracytoplasmic IL-10 expression were quantified with flow cytometry. In basal conditions, CD154 expression was not different in patients and controls. B cell stimulation did not cause a significant increase in CD154 expression in control B cells. However, its expression increased 2 times in B cells obtained from SLE patients. IL-10 expression was confined to CD154(+) cells. Our results show that IL-10 production is intimately linked to CD154 expression in B cells, and that the IL-10(+)CD154(+) B cell subset increases abnormally when SLE-derived cells are stimulated with CSS.

Behcet’s disease is a multi-system inflammatory disorder of unknown etiology. The disease is more prevalent in Eastern Mediterranean countries and Japan where there is a linkage to HLA-B51. Mexican Mestizos are suitable subjects for studying the role of ethnicity in the susceptibility to Behcet’s disease. High-resolution HLA class I and class II typing was performed by polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) reverse dot blot and PCR-single-strand polymorphism in 32 patients with Behcet’s disease and 99 healthy ethnically-matched controls. A significant increased frequency of HLA-B * 44 ( P =0.02; OR = 2.78; CI 95% = 1.1–7.7), HLA-B * 52 ( P =0.02; OR = 5.33; CI 95% = 1.07–29.1), and HLA-B * 56 ( P =0.003; OR = 4.19; CI 95% = 3.37–5.21) as well as HLA-DRB1 * 01 and HLA-DRB1 * 13 ( P =0.007; OR = 3.36; CI 95% = 1.22–9.27) was found in Mexican patients with Behcet’s disease when compared to controls. The low frequency of native markers in Mexican Mestizo patients with Behcet’s disease suggests that genetic admixture between Eastern Mediterraneans and Orientals with Amerindians is a recent event that increased the risk of developing Behcet’s disease in the Mexican population.

CD4+ T cells that constitutively express CD25 exhibit powerful suppressive properties. Such cells have been denominated regulatory T cells (T(R)). Alterations in T(R)cells are known to cause organ-specific autoimmune disease in animal models. The aim of this work was to quantify CD4+CD25+ T cells in patients with systemic lupus erythematosus (SLE). Thirty untreated patients (ten with active disease) and ten healthy volunteers were studied. Flow cytometry was used to quantify cell populations. CD4+CD69+, CD4+CD25+ and CD4+CD25(bright) cells were considered. Peripheral blood mononuclear cell cultures were performed and supernatants collected for IL-10 and 12 measurement. CD4+CD25+ cells were significantly decreased in patients with active disease when compared to control subjects and patients without disease activity (P

Complement activation takes place in autoimmune diseases and accounts for tissue inflammation. Previously, complement inhibition has been considered for the treatment of SLE. Complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the alternative pathway of complement and a soluble form reverses established inflammation and bone destruction in experimental autoimmune arthritis. We asked whether specific inhibition of the alternative pathway could inhibit autoimmunity and/or organ damage in lupus-prone mice. Accordingly, we treated lupus-prone MRL/lpr mice with a soluble form of CRIg (CRIg-Fc) and we found that it significantly diminished skin lesions, proteinuria and pyuria, and kidney pathology. Interestingly, serum levels of anti-DNA antibodies were not affected despite the fact that serum complement 3 (C3) levels increased significantly. Immunofluorescent staining of kidney tissues revealed a reduction in staining intensity for C3, IgG, and the macrophage marker Mac-2. Thus our data show that inhibition of the alternative pathway of complement controls skin and kidney inflammation even in the absence of an effect on the production of autoantibodies. We propose that CRIg should be considered for clinical trials in patients with systemic lupus erythematosus.

Foxp3(+) regulatory T cells (Treg) are pivotal for the maintenance of peripheral tolerance and prevent development of autoimmune diseases. We have reported that calcium/calmodulin-dependent protein kinase IV (CaMK4) deficient MRL/lpr mice display less disease activity by promoting IL-2 production and increasing the activity of Treg cells. To further define the mechanism of CaMK4 on Treg cells in systemic lupus erythematosus (SLE), we used the Foxp3-GFP reporter mice and treated them with KN-93, an inhibitor of CaMK4.We generated MRL/lpr Foxp3-GFP mice to record Treg cells; stimulated naïve CD4(+) T cells from MRL/lpr Foxp3-GFP mice under Treg polarizing conditions in the absence or presence of KN-93; evaluated the number of GFP positive cells in lymphoid organs and examined skin and kidney pathology at 16 weeks of age. We also examined the infiltration of cells and recruitment of Treg cells in the kidney.We show that culture of MRL/lpr Foxp3-GFP T cells in the presence of KN-93 promotes Treg differentiation in a dose-dependent manner. Treatment of MRL/lpr Foxp3-GFP mice with KN-93 results in a significant induction of Treg cells in the spleen, peripheral lymph nodes and peripheral blood and this is accompanied by decreased skin and kidney damage. Notably, KN-93 clearly diminishes the accumulation of inflammatory cells along with reciprocally increased Treg cells in target organ.Our results indicate that KN-93 treatment enhances the generation of Treg cells in vitro and in vivo highlighting its potential therapeutic use for the treatment of human autoimmune diseases.

Epigenetic events play a central role in the priming, differentiation and subset determination of T lymphocytes. Through their influence on chromatin conformation and DNA-accessibility to transcription factors and RNA polymerases, epigenetic marks allow or prevent gene expression and control cellular functions including cytokine expression. CpG-DNA methylation and post-translational modifications to histone tails are the two most well accepted epigenetic mechanisms. The involvement of epigenetic mechanisms in the pathogenesis of systemic lupus erythematosus (SLE) has been suggested by the development of lupus-like symptoms by individuals who received procainamide or hydralazine treatment resulting in a reduction of CpG-DNA methylation. To date, a growing body of literature indicates that the deregulation of cytokine expression through epigenetic disturbances can result in altered immune responses and autoimmune reactions. Over the past decade, various global and regional epigenetic alterations have been reported in immune cells from patients with SLE and other autoimmune disorders. More recently, the molecular mechanisms that result in epigenetic disturbances have been addressed, and deregulated transcription factor networks have been demonstrated to mediate epigenetic alterations in B and T lymphocytes from SLE patients. A better understanding of the molecular events that contribute to epigenetic alterations and subsequent immune imbalance is essential for the establishment of disease biomarkers and identification of potential therapeutic targets.

The activity of calcium/calmodulin-dependent protein kinase IV (CaMK4) is increased in T cells from patients with systemic lupus erythematosus (SLE) and has been shown to reduce IL-2 production by promoting the effect of the transcriptional repressor cAMP responsive element modulator-α on the IL2 promoter. In this article, we demonstrate that T cells from MRL/lpr mice display increased levels of CaMK4 in the nucleus, and that genetic deletion of Camk4 results in improved survival. We demonstrate that absence of CaMK4 restores IL-2 production, curbs increased T cell activation, and augments the number and activity of regulatory T cells. Analogously, silencing of CaMK4 in T cells from patients with SLE increases the expression of FoxP3 on stimulation in the presence of TGF-β. Our results demonstrate the importance of the serine/threonine kinase CaMK4 in the generation and function of regulatory T cells in patients with SLE and lupus-prone mice, and its potential to serve as a therapeutic target.

The contribution of individual molecular aberrations to the pathogenesis of systemic lupus erythematosus (SLE), an autoimmune disease that affects multiple organs, is often difficult to evaluate because of the presence of abundant confounding factors. To assess the effect of increased expression of the phosphatase protein phosphatase 2A (PP2A) in T cells, as recorded in SLE patients, we generated a transgenic mouse that overexpresses the PP2Ac subunit in T cells. The transgenic mouse displays a heightened susceptibility to immune-mediated glomerulonephritis in the absence of other immune defects. CD4+ T cells produce increased amounts of IL-17 while the number of neutrophils in the peripheral blood is increased. IL-17 neutralization abrogated the development of glomerulonephritis. We conclude that increased PP2Ac expression participates in SLE pathogenesis by promoting inflammation through unchecked IL-17 production and facilitating the development of end-organ damage.

Neurocysticercosis (NC) is caused by the establishment of Taenia solium cysticerci in the central nervous system. Previous studies have established that neuroinflammation plays a key role in the severity of the disease. However, the relationship between peripheral and local immune response remains inconclusive. This work studies the peripheral and local immune-inflammatory features and their relationships, toward the identification of potential peripheral immunologic features related to severity. A panel of cytokines was measured in paired cerebrospinal fluid (CSF) and in the supernatant of antigen-specific stimulated peripheral blood mononuclear cells samples (SN) in a total of 31 untreated inflammatory and non-inflammatory NC patients. Increased clinical and radiologic severity was associated with an increased cerebrospinal fluid cell count. A peripheral proliferative depression that negatively correlates with CSF cellularity and TNFα and that positively correlates with SN IL5 was observed in severe NC patients. These results provide evidences to support the systemic proliferative response as a biomarker to monitor the level of neuroinflammation, of possible value in the patients' follow-up during treatment.

Summary Lymphopenia is a common clinical manifestation in patients with systemic lupus erythematosus (SLE). However, its physiopathogenic role and the contribution of different T cell subsets in this setting have not been addressed fully. The aim of this study was to characterize T cell subsets quantitatively and functionally and their association with lymphopenia and azathioprine treatment in SLE. We included 84 SLE patients and 84 healthy controls and selected 20 patients for a 6-month longitudinal analysis. Peripheral blood mononuclear cells were isolated, and T cell subsets were analysed by flow cytometry. Functional analyses included autologous and allogeneic co-cultures of T cells. Our data show persistently lower absolute numbers of CD4+CD25high T cells [regulatory T cells (Tregs)] (1·9 versus 5·2, P

The origin and function of human double negative (DN) TCR-αβ+ T cells is unknown. They are thought to contribute to the pathogenesis of systemic lupus erythematosus because they expand and accumulate in inflamed organs. In this study, we provide evidence that human TCR-αβ+ CD4− CD8− DN T cells can derive from activated CD8+ T cells. Freshly isolated TCR-αβ+ DN T cells display a distinct gene expression and cytokine production profile. DN cells isolated from peripheral blood as well as DN cells derived in vitro from CD8+ T cells produce a defined array of proinflammatory mediators that includes IL-1β, IL-17, IFN-γ, CXCL3, and CXCL2. These results indicate that, upon activation, CD8+ T cells have the capacity to acquire a distinct phenotype that grants them inflammatory capacity.

The regulation of IL-2 production is central to our understanding of the immune system. Key during T cell activation, it also plays an essential role in the regulation of the immune response. This review discusses the function of recently described factors that modulate transcription and chromatin remodeling at the IL2 promoter. Also, it addresses the role of FoxP3 as a transcriptional regulator in conventional T cells and regulatory T cells, and the mechanisms whereby CD28 stabilizes IL2 transcription and translation. Finally, the alterations that prevent T cells from SLE patients from producing normal amounts of IL-2 upon stimulation are described.

Elements of the innate and adaptive immune response have been implicated in the development of tissue damage after ischemic reperfusion (I/R). Here we demonstrate that T cells infiltrate the intestine of C57BL/6 mice subjected to intestinal I/R during the first hour of reperfusion. The intensity of the T cell infiltration was higher in B6.MRL/lpr mice subjected to intestinal I/R and reflected more severe tissue damage than that observed in control mice. Depletion of T cells limited I/R damage in B6.MRL/lpr mice, whereas repletion of B6.MRL/lpr lymph node-derived T cells into the I/R-resistant Rag-1−/− mouse reconstituted tissue injury. The tissue-infiltrating T cells were found to produce IL-17. Finally, IL-23 deficient mice, which are known not to produce IL-17, displayed significantly less intestinal damage when subjected to I/R. Our data assign T cells a major role in intestinal I/R damage by virtue of producing the pro-inflammatory cytokine IL-17.

We discuss a 53-year-old woman with systemic lupus erythematosus who presented with vasculitis, hypocomplementemia and nephritis. Although her serum complement 4 (C4) levels were zero, she had four copies of C4 gene. Renal biopsy revealed membranoproliferative glomerulonephritis and the presence of cryoglobulins, detected by electron microscopy, and significant numbers of T cells in the interstitium. Cryoglobulins were considered responsible for the complete consumption of C4 in the serum the levels of which improved gradually after treatment. T cells in the kidney were found to express CD44 and phosphorylated ezrin/radixin/moiesin which explain why they homed to the kidney inappropriately. The contribution of cryoglobulins and T cells in the expression of kidney pathology is discussed.

T cells from patients with systemic lupus erythematosus exhibit a notable array of defects that probably contribute to the origin and development of the disease. Such abnormalities include an abnormal response to stimulation, aberrant expression of molecules that play key roles in intracellular signalling pathways, altered transcription factor activation and binding, and skewed gene expression. The combination of these alterations leads the cell to the expression of a particular phenotype that intense research has gradually uncovered over the last years. The aim of this article is to review the findings that have allowed us to better understand the behaviour of the lupus T cell and highlight the molecules that represent potential therapeutic targets.

T cells from patients with systemic lupus erythematosus (SLE) exhibit several discrete and specific defects that alter signaling pathways and, thus, the gene expression pattern and behavior upon stimulation. Rewiring of the CD3 complex and aggregation of surface-membrane lipid rafts grant SLE T cells a lower activation threshold and distort the ensuing signaling events. Additionally, increased expression of adhesion molecules within aggregated lipid rafts guides them to target organs. Aberrant cell signaling causes altered transcription factor expression and abnormal DNA-methylation patterns that lead to skewed gene expression. The result is an abnormally functioning T cell that exhibits several molecular alterations that can be exploited as therapeutic or diagnostic markers.

Dendritic cells (DC) play a pivotal role in the development of immune responses and the maintenance of peripheral tolerance. They continuously patrol tissues and sample antigens in search for the presence of inflammatory or pathogen-derived signals; they mature in accordance to signs prevalent in their environment and, by doing so, acquire certain features that will allow them to convey a precise message — either productive or regulatory — to the T cells they will encounter within regional lymph nodes. Evidence suggests that DC are involved in the pathogenesis of SLE. Their capacity to induce an antinuclear-oriented autoimmune response has been proven in animal models. Further, their maturation process is abnormal and their cytokine secretion and T cell stimulation is biased in patients with SLE. Lupus, however, is a problematic environment for their study and characterization. Sera from SLE patients contain several factors capable of inducing phenotypic and functional changes in DC. Thus, the abnormalities that have been reported may represent DC-intrinsic defects, a skewed phenotype due to an abnormal milieu, or a combination of both. In this review we discuss the available information in an attempt to reconcile these issues.

Background IL-17 producing T helper (Th17) cells have been closely associated with the development of organ damage in inflammatory and autoimmune diseases. We have previously reported that Calcium/calmodulin-dependent protein kinase IV (CaMK4) plays a crucial role in Th17 cell differentiation invitro and invivo and suggested that CaMK4 inhibition ameliorates clinical and pathological findings in experimental autoimmune encephalomyelitis (EAE) and MRL/lpr mice (1). We have also shown that Il-17 is important in the expression of anti-glomerular basement membrane Ab-induced glomerulonephritis (AIGN) (2). However, the effect of CaMK4 on recruitment of pathogenic T cells to target tissues in inflammatory settings has not been studied. Objectives The aim of this study is to determine the role of CaMK4 in the infiltration of inflammatory cells to target tissues and to explore its mechanism. Methods We induced experimental AIGN in Camk4 sufficient or deficient mice and compared the kidney injury including the number of IL-17 producing cells and the expression of CCR6 and CCL20 in both groups. We also evaluated the effect of KN-93, a compound of CaMK4 antagonist in this AIGN model. Results Camk4 deficient mice displayed less glomerular injury and less proteinuria at day 14 and at day 21 after induction of AIGN. The expression of CCR6 and CCL20 in inflamed kidney is lower in Camk4 deficient mice. Kidney infiltration by Th17 producing CD4+ T cells was decreased significantly in Camk4 deficient mice, suggesting that CaMK4 facilitated AIGN damage by promoting local inflammatory cells accumulation. In line with these observations, KN-93 treatment improved clinical and pathological finings in mice induced AIGN at dose dependent manner. Conclusions Collectively, our results indicate that CaMK4 inhibition might be a novel therapeutic strategy of Th17 cells-mediated inflammatory diseases. References CaMK4-dependent activation of AKT/mTOR and CREM-α underlies autoimmunity-associated Th17 imbalance. Koga T, Hedrich CM, Mizui M, Yoshida N, Otomo K, Lieberman LA, Rauen T, Crispin JC, Tsokos GC. J Clin Invest. 2014 May 1;124(5):2234-45. Cutting edge: protein phosphatase 2A confers susceptibility to autoimmune disease through an IL-17-dependent mechanism.Crispin JC, Apostolidis SA, Rosetti F, Keszei M, Wang N, Terhorst C, Mayadas TN, Tsokos GC.J Immunol. 2012 Apr 15;188(8):3567-71. Disclosure of Interest None declared

The aim of this work was to discuss the current knowledge concerning regulatory T cells in the pathogenesis of autoimmune diseases. CD4 + T cells that constitutively express CD25 exhibit powerful suppressive properties. Such cells have been denominated regulatory T cells (T R ). Alterations in T R cells are known to cause organ-specific autoimmune disease in animal models. These cells are anergic when stimulated via their TCR but proliferate when costimulated with IL-2. A particular characteristic is that CD4 + CD25 + T cells inhibit the proliferative responses of CD4 + CD25 − T cells by suppressing the capacity of the responders to transcribe IL-2. The survival and/or expansion of this regulatory subset in the periphery appears to need the availability of IL-2, the components of the IL-2R, as well as cell surface costimulatory molecules. Cytokine participation has been shown in many of the in vivo models of autoimmunity where regulatory cells participate, providing evidence in favour of a role for IL-10, transforming growth factor-β and IL-4. The behavior and possible participation of regulatory T cells in human disease is still a poorly explored topic but their pathogenic role is warranted.

Previously it was shown that the TNF superfamily member TWEAK (TNFSF12) acts through its receptor, Fn14, to promote proinflammatory responses in kidney cells, including the production of MCP-1, RANTES, IP-10 and KC. In addition, the TWEAK/Fn14 pathway promotes mesangial cell proliferation, vascular cell activation, and renal cell death. To study the relevance of the TWEAK/Fn14 pathway in the pathogenesis of antibody-induced nephritis using the mouse model of nephrotoxic serum nephritis (NTN), we induced NTN by passive transfer of rabbit anti-glomerular antibodies into Fn14 knockout (KO) and wild type (WT) mice. Severe proteinuria as well as renal histopathology were induced in WT but not in Fn14 KO mice. Similarly, a pharmacologic approach of anti-TWEAK mAb administration into WT mice in the NTN model significantly ameliorated proteinuria and improved kidney histology. Anti-TWEAK treatment did not affect the generation of mouse anti-rabbit antibodies; however, within the kidney there was a significant decrease in glomerular immunoglobulin deposition, as well as macrophage infiltrates and tubulointerstitial fibrosis. The mechanism of action is most likely due to reductions in downstream targets of TWEAK/Fn14 signaling, including reduced renal expression of MCP-1, VCAM-1, IP-10, RANTES as well as Fn14 itself, and other molecular pathways associated with fibrosis in anti-TWEAK treated mice. Thus, TWEAK/Fn14 interactions are instrumental in the pathogenesis of nephritis in the NTN model, apparently mediating a cascade of pathologic events locally in the kidney rather than by impacting the systemic immune response. Disrupting TWEAK/Fn14 interactions may be an innovative kidney-protective approach for the treatment of lupus nephritis and other antibody-induced renal diseases.

Background Autoimmune diseases such as systemic lupus erythematosus (SLE) display quantitative (1) and/or qualitative (2) deficiencies of Foxp3+ regulatory T (Treg) cells and this is considered to contribute to immune dysregulation and organ damage (3). Calcium/calmodulin-dependent protein kinase IV (CaMK4) is expressed at increased levels in T cells from SLE patients (4) and lupus-prone mice (5). Objectives Treg are pivotal for the maintenance of peripheral tolerance and prevent development of autoimmune diseases. We have reported that CaMK4 deficient MRL/ lpr mice display less disease activity by promoting IL-2 production and increasing the activity of Treg cells. To further define the mechanism of CaMK4 on Treg cells in SLE, we used the Foxp3 -GFP reporter mice and treated them with KN-93, an inhibitor of CaMK4. Methods We generated MRL/ lpr Foxp3- GFP mice to record Treg cells; stimulated naive CD4+ T cells from MRL/ lpr Foxp3- GFP mice under Treg polarizing conditions in the absence or presence of KN-93; evaluated the number of GFP` positive cells in lymphoid organs and examined skin and kidney pathology at 16 week of age. We also examined the infiltration of cells and recruitment of Treg cells in the kidney. Results We show that culture of MRL/ lpr Foxp3- GFP T cells in the presence of KN-93 promotes Treg differentiation in a dose dependent manner (Fig. F). Treatment of MRL/ lpr Foxp3- GFP mice with KN-93 results in significant induction of Treg cells in the spleen, peripheral lymph nodes (Fig. B-E) and peripheral blood (Fig. A and B) and this is accompanied by decreased skin and kidney damage. Notably, KN-93 clearly diminishes the accumulation of inflammatory cells along with reciprocally increased Treg cells in target organ. ![Figure 1][1] Figure 1 Conclusions Our results indicate that KN-93 treatment enhances the generation of Treg cells in vitro and in vivo highlighting its potential therapeutic use for the treatment of human autoimmune diseases. References 1. Valencia X, Yarboro C, Illei G, Lipsky PE. Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus. J Immunol. 2007;178(4):2579-88. 2. Yan B, Ye S, Chen G, Kuang M, Shen N, Chen S. Dysfunctional CD4+,CD25+ regulatory T cells in untreated active systemic lupus erythematosus secondary to interferon-alpha-producing antigen-presenting cells. Arthritis Rheum. 2008;58(3):801-12. 3. Shevach EM. Mechanisms of foxp3+ T regulatory cell-mediated suppression. Immunity. 2009;30(5):636-45. 4. Juang YT, Wang Y, Solomou EE, Li Y, Mawrin C, Tenbrock K, et al. Systemic lupus erythematosus serum IgG increases CREM binding to the IL-2 promoter and suppresses IL-2 production through CaMKIV. J Clin Invest. 2005;115(4):996-1005. 5. Koga T, Ichinose K, Mizui M, Crispin JC, Tsokos GC. Calcium/Calmodulin-Dependent Protein Kinase IV Suppresses IL-2 Production and Regulatory T Cell Activity in Lupus. J Immunol. 2012;189(7):3490-6. Acknowledgements This work was supported by NIH grants RO1 AI R01AI085567 and AR064350. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.2491 [1]: pending:yes