Inhibiting TLR7 Expression in the Retinal Pigment Epithelium Suppresses Experimental Autoimmune Uveitis

Experimental autoimmune uveitis (EAU), a model of human uveitis, is an organ-specific, T cell-mediated autoimmune disease. Autoreactive T cells can penetrate the blood-retinal barrier, which is a physical defense composed of tight junction-linked retinal pigment epithelial (RPE) cells. RPE cells serve as antigen-presenting cells (APCs) in the eye since they express MHC class I and II and Toll-like receptors (TLRs). Although previous studies have shown that supplementation with TLR agonists exacerbates uveitis, little is known about how TLR signaling in the RPE contributes to the development of uveitis. In this study, we isolated the RPE from EAU mice, which were induced by active immunization (aEAU) or adoptive transfer of antigen-specific T cells (tEAU). The expression of TLRs on RPE was determined, and both aEAU and tEAU mice exhibited induced tlr7 expression. The TLR7 agonist R848 was shown to induce aggressive disease progression, along with significantly elevated levels of the uveopathogenic cytokine IL-17. Furthermore, not only IL-17 but also R848 appeared to enhance the inflammatory response and to impair the barrier function of the RPE, indicating that TLR7 signaling is involved in the pathogenesis of EAU by affecting the behaviors of the RPE and consequently allowing the infiltration of autoreactive T cells intraocularly. Finally, local application of shRNA against TLR7 delivered by recombinant AAV effectively inhibited disease severity and reduced IFN-γ and IL-17. Our findings highlight an immunomodulatory role of RPE TLR7 in EAU development and provide a potential therapeutic strategy for autoimmune uveitis.

CD138 Expression Promotes Accumulation and Activation of T Cells in Autoimmune MRL/lpr Mice

CD138+ T cells that accumulated in Fas-deficiency lupus mice, had been identified as autoreactive T cells in SLE which significantly promoted autoantibody secretion. In present study, we found CD138 expression in T cells played a key role in the progression of SLE in MRL/lpr mice. Our results indicated CD138+ T cells apoptosis was in Fas dependent way. However, CD138 expression of T cells in MRL/lpr mice could significantly prevent T cells apoptosis, contribute to accumulation of T cells and DN T cells and simultaneously promote T cells activation. Importantly, CD138 expression in DN T cells significantly increased FasL expression of DN T cells enhancing the cytotoxity of DN T cells. Phorbol 12-myristate 13-acetate and Ionomycin (PI) stimulation could significantly prevent CD138+ T cells accumulation by strikingly inducing their specific apoptosis. Moreover, PI stimulation significantly activated CD138+ T cells with increased CD69 expression in them. Importantly, our results showed CD69 expression in CD138+ T cells could significantly increase the apoptosis level of them. That indicated PI stimulation could induce specific apoptosis of CD138+ T cells via increasing CD69 expression in CD138+ T cells. In addition, our results showed CD138- T cells in MRL/lpr mice had significant defects in activation. However, to activate T cells could significantly prevent CD138 expression in CD3+ T cells of MRL/lpr mice. Our results suggested CD138 expression in CD3+ T cells of MRL/lpr mice was probably caused by the failure of activation in autoreactive T cells before self-antigens exposure to immune system.

Peripheral tolerance by Treg via constraining OX40 signal in autoreactive T cells against desmoglein 3, a target antigen in pemphigus

Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor–transgenic mice to thymus-transplanted mice, Dsg3-specific CD4+ T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3−/− mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance.

Predictors of Efficacy in Chinese Patients with Severe Aplastic Anemia Treated with Eltrombopag and Intensive Immunosupressive Therapy

Prospective trials showed the clinical efficacy of eltrombopag in refractory/relapsed aplastic anemia (AA), with up to 40% hematologic improvement [1]. Moreover, eltrombopag was combined with frontline immunosupressive therapy (IST) consisited of antithymocyte immunoglobulin (ATG) and cyclosporin (CsA), with an overall response rate exceeding 80% [2]. The metabolism of eltrodopag is different in disparate population. Currently, the recommended dose is 75 mg/d for East Asian populations [2], Pretreatment clinical and laboratory characteristics predicting eltrombopag response are still unclear in severe AA (SAA) patients of real-world in East Asian. From May 2014 to December 2020, 59 adult SAA patients were treated with IST (r-ATG, 3.5mg/kg/d × 5d and CsA 3-5 mg/kg/d) in conjunction with eltrodopag at the dose of 75 mg/d from day 1 until 6 months. We used Receiver Operating Characteristic (ROC) curve to predict the factors of the efficacy of eltrombopag. Median follow-up was 13 months (6 month to 29 months). Overall 45 patients (76%) responded to eltrombopag, including 16 patients (27%) who achieved complete response (CR) and 29 patients (49%) who achieved partial response (PR), whist 14 patients were non-remission (NR) according to the criteria of the European Group for Blood and Marrow Transplantation (EBMT) [3]. The severity (P=0.032), infection (P=0.039) and lymphocyte count (P=0.021), as well as reticulocyte percentage (P=0.002) were different significantly between groups before the treatment (Table 1). Yet, the median age (38 years and 50.5 years) and median time from diagnosis to treatment (16 d and 27.5 d) are comparable. There was no significant difference in the count of platelet and T cell subsets of peripheral blood. ROC curve was used as one model to predict factors linked to the effects of eltrodopag in 6 months. The lymphocyte count (P=0.042, AUC=0.721, 95%CI 0.537-0.904), the percentage of reticulocyte (P=0.007, AUC=0.793, 95%CI 0.631-0.955), and red cell distribution width - coefficient of variation (RDW-CV) (P=0.042, AUC=0.721, 95%CI 0.493-0.948) were positively correlated with the efficacy of eltrombopag (Table 2, Figure 1). Discussion It has been reported that moderate diseaseand lymphoid marrow infiltration were related with the response to eltrombopag in older AA patients [4]. Scheinberg et al. has showed that predictive factors related to IST of 6th month were including the age, lymphocyte count and reticulocyte count [5]. We found that the lymphocyte count, the percentage of reticulocytes and RDW-CV were related with response. The increase of RDW-CV is related to production of reticulocyte. A higher lymphocyte count, the percentage of reticulocytes and RDW-CV may indicate better residual marrow function. Lymphocyte count is also associated with abnormal immunity. Hematopoietic recovery of SAA depends on IST involving elimination of autoreactive T cells that target progenitor cells in the bone marrow, and sufficient residual stem cells to support blood cell production after IST. IST combined with eltrombopag could eradicate autoreactive T cells and promote hematopoiesis simultaneously, thereby, the relevant indicators for predicting efficacy also reflect this related status. This study was limited by its retrospective nature, small cohorts, an absence of randomization a treatment comparator, and IST was based on r-ATG and CsA. In preliminar conclusion, the pretreatment lymphocyte count, the percentage of reticulocytes and RDW-CV together serve as a simple predictor of response following IST combined with eltrombopag for Chinese patients with SAA. Reference [1] Olnes MJ, et al. Eltrombopag and improved hematopoiesis in refractory aplastic anemia. N Engl J Med. 2012;367(3):11-19. [2] Townsley DM, et al. Eltrombopag Added to Standard Immunosuppression for Aplastic Anemia [J]. N Engl J Med, 2017, 376 (16): 1540-1550. [3] de Latour, RP, et al. Results of the EBMT SAAWP Phase III Prospective Randomized Multicenter RACE Study of Horse ATG and Ciclosporin with or without Eltrombopag in naive SAA patients. EBMT 2020 abstract O018. [4] Fattizzo B, et al. Clinical and morphological predictors of outcome in older aplastic anemia patients treated with eltrombopag. Haematologica. 2019 Nov;104(11): e494-e496. [5] Scheinberg P,et al. Predicting response to immunosuppressive therapy and survival in severe aplastic anaemia. Br J Haematol. 2009 Jan;144(2):206-16. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Pathologic Factors and Spectrum of Central Nervous System Inflammatory Demyelinating Diseases

Multiple sclerosis (MS) is the most common cause of nontraumatic disability in young adults. It is a chronic inflammatory demyelinating disease of the central nervous system. Traditionally, MS has been considered an autoimmune disorder consisting of myelin autoreactive T cells that drive an inflammatory process, leading to secondary macrophage recruitment and subsequent myelin destruction. However, accumulating data based on increasing numbers of probes that can be effectively applied to MS tissue have indicated that the events involved in the immunopathogenesis of MS may be more complicated.

Thymic B Cells as a New Player in the Type 1 Diabetes Response

Type 1 diabetes (T1d) results from a sustained autoreactive T and B cell response towards insulin-producing β cells in the islets of Langerhans. The autoreactive nature of the condition has led to many investigations addressing the genetic or cellular changes in primary lymphoid tissues that impairs central tolerance- a key process in the deletion of autoreactive T and B cells during their development. For T cells, these studies have largely focused on medullary thymic epithelial cells (mTECs) critical for the effective negative selection of autoreactive T cells in the thymus. Recently, a new cellular player that impacts positively or negatively on the deletion of autoreactive T cells during their development has come to light, thymic B cells. Normally a small population within the thymus of mouse and man, thymic B cells expand in T1d as well as other autoimmune conditions, reside in thymic ectopic germinal centres and secrete autoantibodies that bind selective mTECs precipitating mTEC death. In this review we will discuss the ontogeny, characteristics and functionality of thymic B cells in healthy and autoimmune settings. Furthermore, we explore how in silico approaches may help decipher the complex cellular interplay of thymic B cells with other cells within the thymic microenvironment leading to new avenues for therapeutic intervention.

Cellular Mechanism of Immunology in Systemic Lupus Erythematosus

To date, the mechanism of systemic lupus erythematosus (SLE) has not been thoroughly deciphered. Recent research demonstrated that CD138+ T cells accumulate in an SLE murine model, indicating that they are autoreactive T cells that significantly promote autoantibody production. Double negative (DN) T cells have been demonstrated to participate in the progression of SLE, but their detailed mechanism and the role in SLE remain unclear. Importantly, the expression of CD138 in CD3+ T cells plays a key role in the progression of lupus; it causes the accumulation of autoreactive T cells, including DN T cells, by significantly preventing their apoptosis. T helper 1 cells and interferon gamma both prevail in SLE; they may play essential roles in building the inflammatory condition of SLE. Defects occur in regulatory B (Breg) cells during their expansion in SLE, resulting in more differentiation of activated B cells into plasma cells; this subsequently increases antibody production. Myeloid-derived suppressor cells (MDSCs) enhance the expansion of Breg cells. However, the sustained increase of cytokine levels in SLE promotes the differentiation of more MDSCs into macrophage and dendritic cells, resulting in the defective expansion of MDSCs. The defective expansion of Breg cells and MDSCs breaks the immune-tolerance milieu in SLE, resulting in increased autoantibody secretion from those abnormal plasma cells. This review discusses recent advances regarding the detailed roles and mechanisms of these immunocytes in SLE.

Hyperbaric Oxygen Therapy Alleviates the Autoimmune Encephalomyelitis via the Reduction of IL-17a and GM-Csf Production of Autoreactive T Cells as Well as Boosting the Immunosuppressive IL-10 in the Central Nervous System Tissue Lesions

Multiple sclerosis (MS) is a chronic autoimmune disease mainly caused by autoreactive T cells, followed by neuronal demyelination and disabling paralysis. Hyperbaric oxygen therapy (HBOT) is usually an adjunct to therapy for the treatment of neurological disorders. However, it remains still controversial whether HBOT is an effective option for the treatment of MS. Experimental autoimmune encephalomyelitis (EAE) is a well-studied mouse model investigated for the MS pathogenesis and the efficacy of the therapeutic intervention. Both encephalitogenic Th1 and Th17 are pivotal T cell subsets immunopathogenically producing several disease-initiating/modifying cytokines in the central nervous system (CNS) lesions to further exacerbate/ameliorate the progression of EAE or MS. However, it remains unclear whether HBOT modulates the context of T helper cell subsets in CNS lesions. We employed EAE in the presence of HBOT to assess whether disease amelioration is attributed to alterations of CNS-infiltrating T cell subsets. Our results demonstrated that semi-therapeutic HBOT significantly alleviated the progression of EAE, at least, via the suppression of Th17 response, the downregulation of CD4 T helper cells expressing GM-CSF or TNF-α, and the boosting of immunomodulatory IL-4 or IL-10-expressed CD4 T cells in the CNS lesions. Conclusively, HBOT attenuated EAE through the modulation of T cell responses in an earlier stage.