Breakthrough treatment for refractory and relapsed immune thrombocytopenia (ITP) patients is urgently needed. Autoantibody- mediated platelet clearance and megakaryocyte dysfunction are important pathogenic mediators of ITP. Glycoprotein (GP) Ibα is a significant autoantigen found in ITP patients and is associated with poor response to standard immunosuppressive treatments. Here, we engineered human T cells to express a chimeric autoantibody receptor (CAAR) with GPIbα constructed into the ligand-binding domain fused to the CD8 transmembrane domain and CD3ζ-4-1BB signaling domains. We performed cytotoxicity assays to assess GPIbα CAAR T-cell selective cytolysis of cells expressing anti-GPIbα B-cell receptors in vitro. Furthermore, we demonstrated the potential of GPIbα CAAR T cells to persist and precisely eliminate GPIbα-specific B cells in vivo. In summary, we present a proof of concept for CAAR T-cell therapy to eradicate autoimmune B cells while sparing healthy B cells with GPIbα CAAR T cells that function like a Trojan horse. GPIbα CAAR T-cell therapy is a promising treatment for refractory and relapsed ITP patients.

Despite diverse therapeutic options for immune thrombocytopaenia (ITP), drug efficacy and selection challenges persist. This study systematically identified potential indicators in ITP patients and followed up on subsequent treatment. We initially analysed 61 variables and identified 12, 14, and 10 candidates for discriminating responders from non-responders in glucocorticoid (N = 215), thrombopoietin receptor agonists (TPO-RAs) (N = 224), and rituximab (N = 67) treatments, respectively. Patients were randomly assigned to training or testing datasets and employing five machine learning (ML) models, with eXtreme Gradient Boosting (XGBoost) area under the curve (AUC = 0.89), Decision Tree (DT) (AUC = 0.80) and Artificial Neural Network (ANN) (AUC = 0.79) selected. Cross-validated with logistic regression and ML finalised five variables (baseline platelet, IP-10, TNF-α, Treg, B cell) for glucocorticoid, eight variables (baseline platelet, TGF-β1, MCP-1, IL-21, Th1, Treg, MK number, TPO) for TPO-RAs, and three variables (IL-12, Breg, MAIPA-) for rituximab to establish the predictive model. Spearman correlation and receiver operating characteristic curve analysis in validation datasets demonstrated strong correlations between response fractions and scores in all treatments. Scoring thresholds SGlu ≥ 3 (AUC = 0.911, 95% CI, 0.865-0.956), STPO-RAs ≥ 5 (AUC = 0.964, 95% CI 0.934-0.994), and SRitu = 3 (AUC = 0.964, 95% CI 0.915-1.000) indicated ineffectiveness in glucocorticoid, TPO-RAs, and rituximab therapy, respectively. Regression analysis and ML established a tentative and preliminary predictive scoring model for advancing individualised treatment.

BACKGROUND:This study aims to explore the potential mechanism of action of the newly discovered hsa_circ_0128899 (circSPEF2) in diffuse large B-cell lymphoma (DLBCL). METHODS:circSPEF2, miR-16-5p and BTB and CNC homologue 2 (BACH2) expression patterns in DLBCL patients and cell lines were studied by RT-qPCR. The biological function of circSPEF2 in vitro and in vivo was investigated by function acquisition experiments. The proliferation activity of lymphoma cells was detected by MTT. Bax, Caspase-3, and Bcl-2 were determined by Western Blot. Apoptosis and the ratio of CD4 to Treg of immune cells in the co-culture system were analyzed by flow cytometry. The mechanism of action of circSPEF2 in DLBCL progression was further investigated by RIP and dual luciferase reporter experiments. RESULTS:circSPEF2 was a circRNA with abnormally down-regulated expression in DLBCL. Increasing circSPEF2 expression inhibited the proliferative activity and induced apoptosis of lymphoma cells in vitro and in vivo, as well as increased CD4+T cells and decreased Treg cell proportion of immune cells in the tumor microenvironment. Mechanically, circSPEF2 was bound to miR-16-5p expression, while BACH2 was targeted by miR-16-5p. circSPEF2 overexpression-mediated effects on lymphoma progression were reversible by upregulating miR-16-5p or downregulating BACH2. CONCLUSIONS:circSPEF2 can influence DLBCL progression by managing cellular proliferation and apoptosis and the proportion of immune cells Treg and CD4 through the miR-16-5p/BACH2 axis.

Uncontrolled inflammation is a pathological state that underlies many diseases. Despite the development of numerous anti-inflammatory agents, the treatment of uncontrolled inflammation remains a challenging task. We developed a targeted delivery system for [5-(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1), a potent inhibitor of the NF-κB signaling pathway. The system comprises TPCA-1-loaded nanoparticles (NPs) functionalized with a monoclonal antibody (mAb) that specifically binds to the break point of the IgD6 region of the platelet/endothelial cell adhesion molecule-1 (PECAM-1) extracellular segment that is overexposed on the injured endothelium and activated macrophages during the pathogenesis of inflammation. In vitro binding and cellular uptake experiments revealed that the mAb modification on NPs could significantly enhance uptake by both Raw264.7 and HUVEC compared with unmodified NPs. In studies conducted at the cellular level focusing on anti-inflammatory and antioxidant effects, this formulation was found to effectively inhibit M1 polarization of macrophages, downregulate the secretion of pro-inflammatory cytokines, and reduce the production of reactive oxygen species (ROS) and nitric oxide (NO). In an animal model of vascular endothelial injury with acute inflammation, these NPs were capable of delivering TPCA-1 to inflammatory lesions in a targeted manner. Compared with the free agent-treated group, the NP-treated group exhibited reduced infiltration of inflammatory cells. In conclusion, our study demonstrates that this targeted delivery of TPCA-1-loaded NPs represents a promising strategy for improved mitigation of uncontrolled inflammation.

Background: Immune thrombocytopenia (ITP) is a bleeding disorder caused by autoantibodies against platelet glycoproteins (GPs). A key attempt to treat ITP is to eliminate the autoreactive B cells. Here, we proposed a new strategy for B cell-depletion therapy of ITP, chimeric autoantibody receptors T cells (CAAR-T), which could trap autoreactive B cells and perform specific killing. GPIbα is a significant autoantigen found in ITP patients and indicates a poor response to standard immunosuppressive treatments. We developed GPIbα-based CAARs in this study. Aim: To precisely eliminate the autoreactive B cells to treat ITP. Methods: We constructed various lengths of the native GPIbα ectodomain into the ligand-binding domain of a second-generation CAR structure and expressed the CAAR structure on the HEK293 cell surface to verify its conformational epitope. We immunized BALB/C mice with human platelet lysates to obtain anti-GPIbα hybridomas and performed a binding assay to confirm selected hybridoma antibodies' binding ability to GPIbα CAAR. We performed cytotoxicity assays to assess GPIbα CAAR-T-cell selective cytolysis against anti-GPIbα B cells in vitro and developed a xenograft model to assess the cytolytic efficiency and security of GPIbα CAAR-T cells in vivo. A plasma antibody binding assay was used to test GPIbα CAAR-T cells' ability in reaction with native GPIbα autoantibodies from ITP patients. We applied an enzyme-linked immunospot (ELISpot) assay to verify GPIbα CAAR-T cells' potential in eradicating autoreactive B cells of ITP patients. Results: We successfully expressed GPIbα CAAR structure on the cell surface and proved that the conformational epitope of GPIbα CAAR is consistent with that of the native human platelet GPIbα. The Schematic of GPIbα CAAR structure is presented in Figure 1A. By immunizing BALB/C mice with human platelet lysates, we obtained four anti-GPIbα hybridomas with different CAAR binding sites. Vitro cytotoxicity assays confirmed GPIbα CAAR-T-cell selective cytolysis of anti-GPIbα hybridoma cells. In a hybridoma xenograft model, we verified that the CAAR-T cells could persist and selectively lyse autoreactive B cells and subsequently reduce autoantibody titers without apparent off-target organ toxicity. Through the plasma antibody binding assay, we demonstrated that GPIbα CAAR could react with sera from ITP patients with anti-GPIbα antibodies (Figure 1B). ELISpot analysis showed that GPIbα CAAR-T cells successfully eliminated anti-GPIbα B cells from a patient with refractory ITP, and normal B cells were not destroyed compared to anti-CD19 CAR-T cells. Conclusion: Our study provides a proof of concept for CAAR-T cell therapy to eradicate autoimmune B cells while sparing healthy B cells, which would compensate for the defects of traditional B cell depletion therapy in ITP. GPIbα CAAR-T therapy would be a promising treatment for refractory and relapsed ITP patients.

Chimeric antigen receptor T (CAR-T) cell therapy is an attractive strategy for patients with relapsed or refractory hematological malignancies including multiple myeloma (MM). T cells are engineered to attack malignant cells that express tumor-associated antigens and better efficacy could be achieved. However, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and hematologic toxicity are still challenges for CAR-T cell therapy. Among them, hematologic toxicity including thrombocytopenia has a longer duration and lasting effect during and after the treatment for some patients. Here, we present 3 cases of hematologic toxicity manifested as refractory thrombocytopenia with platelet autoantibodies positive and plasma thrombopoietin (TPO) concentration elevated after bispecific CAR-T cell therapy in relapsed/refractory (R/R) MM patients who were successfully treated with standard therapy of immune thrombocytopenia (ITP). Without clear pathogenesis or guidance on therapy published, our cases provide a reference for the treatment of thrombocytopenia after CAR-T cell therapy and inspire exploration of the underlying pathophysiological mechanisms.