CD38 Upregulation Research Articles

NIR stimulated epigallocatechin gallate loaded polydopamine with enhanced antibacterial and ROS scavenging abilities for improved infectious wound healing

Infectious wound healing is complicated with and limited by infection and oxidative stress at the wound site. In recent years, various evidences suggest that nanozymes with multiple enzymatic activities have enabled the development of novel strategies for infectious wound healing. In this study, epigallocatechin gallate loaded polydopamine (P@E) was developed to act as a potent reactive oxygen species (ROS) scavenger for scavenging ROS, alleviating inflammatory responses, and promoting infectious wound healing. Combining with near infrared (NIR) irradiation, P@E presented excellent antibacterial ability of Escherichia coli (E. coli, 93.6%) and methicillin-resistant Staphylococcus aureus (MRSA, 87.6%). Specifically, P@E+NIR exhibited the most potent antioxidant, anti-inflammatory and cell proliferation behaviors through down-regulating intracellular ROS levels (81.9% and 94.3% for NIH3T3 and RAW264.7 respectively) and inducible nitric oxide synthase (iNOS) expression level (55.7%), and up-regulating the expression levels of arginase-1 (Arg-1, 71.4%), heat shock protein 70 (HSP70, 48.6%) and platelet endothelial cell adhesion molecule (CD31, 35.3%) compared to control group. Meanwhile, it also efficiently induced M2 directional polarization of lipopolysaccharide induced murine macrophages to achieve anti-inflammation, indicated by the down-regulation of CD86 (86.2%), and up-regulation of CD206 (85.6%). Significantly, it was also observed that P@E+NIR presented the excellent behaviors of inhibiting wound infection, alleviating wound inflammation, as well as promoting skin tissue repairing. Altogether, it has developed the strategy of using P@E combining with NIR irradiation for the synergistic enhanced healing of infectious skin wound, which can serve as a promising therapeutic strategy for its clinical treatment.

5-aminolevulinic acid photodynamic therapy for chronic wound infection in rats with diabetes

Recent research indicated that ulcers and peripheral vascular disease resulting from drug-resistant bacterial infections are the main causes of delayed healing in chronic diabetic wounds. 5-Aminolevulinic acid (ALA) is a second-generation endogenous photosensitizer. The therapeutic effect and mechanism of ALA-mediated photodynamic therapy (ALA-PDT) on methicillin-resistant Staphylococcus aureus (MRSA)-infected wounds in diabetic rats were investigated in this study. The results revealed the promising antibacterial effects of ALA-PDT MRSA in vitro, with a minimum inhibitory concentration and minimum bactericidal concentration of 250 and 500 μM, respectively. ALA-PDT also changed the permeability and structural integrity of bacterial cell membranes by producing reactive oxygen species. Meanwhile, ALA-PDT accelerated wound healing in MRSA-infected diabetic rats, with 5 % ALA-PDT achieving complete sterilization in 14 days and wound closure in 21 days. Treatment with 5 % ALA-PDT additionally improved the histopathological appearance of skin tissue, as well as fibrosis, inflammatory cytokine release, and angiogenesis-related protein expression. These findings indicated that ALA-PDT significantly promoted the healing of MRSA-infected wounds in diabetic rats by eliminating bacteria, inhibiting inflammation, generating granulation tissues, promoting neovascularization, and restoring damaged nerves. In addition, the healing mechanism was related to the activation of inflammatory and angiogenesis pathways through the regulation of tumor necrosis factor-alpha and interleukin-6 expression and upregulation of CD206, CD31, and VEGF. These findings underscored the potential role of ALA-PDT in promoting the healing of chronic diabetic wounds.

An NAD+-dependent metabolic checkpoint regulates hematopoietic stem cell activation and aging.

How hematopoietic stem cells (HSCs) maintain metabolic homeostasis to support tissue repair and regeneration throughout the lifespan is elusive. Here, we show that CD38, an NAD+-dependent metabolic enzyme, promotes HSC proliferation by inducing mitochondrial Ca2+ influx and mitochondrial metabolism in young mice. Conversely, aberrant CD38 upregulation during aging is a driver of HSC deterioration in aged mice due to dysregulated NAD+ metabolism and compromised mitochondrial stress management. The mitochondrial calcium uniporter, a mediator of mitochondrial Ca2+ influx, also supports HSC proliferation in young mice yet drives HSC decline in aged mice. Pharmacological inactivation of CD38 reverses HSC aging and the pathophysiological changes of the aging hematopoietic system in aged mice. Together, our study highlights an NAD+ metabolic checkpoint that balances mitochondrial activation to support HSC proliferation and mitochondrial stress management to enhance HSC self-renewal throughout the lifespan, and links aberrant Ca2+ signaling to HSC aging.

Impact of LAG-3/FGL1 pathway on immune evasive contexture and clinical outcomes in advanced urothelial carcinoma

BackgroundAnti-programmed death-1 (PD-1)/anti-PD-ligand-1 (PD-L1) pathway inhibition is a standard regimen for advanced urothelial carcinoma (UC); however, its limited efficacy has been reflected in reported medium response rates. This study explored...

Synergistic upregulation of PD‑L1 in tumor cells and CD39 in tumor‑infiltrating CD8+ T cells leads to poor prognosis in patients with hepatocellular carcinoma.

The immune escape of tumor cells and functional status of tumor-infiltrating T cells may serve pivotal roles in the tumor immune microenvironment and progression of hepatocellular carcinoma (HCC). The present study enrolled 91 patients with HCC and examined programmed cell death ligand 1 (PD-L1) expression in tumor cells and CD39 expression in tumor-infiltrating CD8+ T cells in patient samples using multiplex immunofluorescence assays. The impact of PD-L1 and CD39 expression levels on the prognosis of patients with HCC was investigated utilizing Kaplan-Meier analyses. The individual upregulation of PD-L1 in tumor cells, as well as the individual upregulation of CD39 expression in tumor-infiltrating CD8+ T cells did not significantly affect the prognosis of patients with HCC. However, the simultaneous upregulation of both PD-L1 in tumor cells and CD39 in tumor-infiltrating CD8+ T cells was associated with reduced overall survival in patients with HCC. Therefore, the results of the present study suggested that the interplay between tumor cell immune escape and tumor-infiltrating immune cell functional status within the tumor immune microenvironment may have had a substantial impact on the prognosis of patients with HCC. Mechanistically, increased expression levels of PD-L1 in tumor cells may improve the immune escape capacity of tumors, whilst upregulation of CD39 in tumor-infiltrating T cells may be associated with T cell exhaustion. Therefore, the upregulation of PD-L1 expression in tumor cells, in conjunction with the exhaustion of tumor-infiltrating CD8+ T cells, could serve as a future potential prognostic indicator of patients with HCC.

Changes in T-cell subsets occur in interstitial lung disease and may contribute to pathology via complicated immune cascade.

The study aimed to investigate the expression profiles of transcription factors, cytokines, and co-stimulatory molecules in helper T (Th)-cell subsets within bronchoalveolar lavage (BAL) samples of patients with interstitial lung diseases (ILDs). Twenty ILDs patients were included in the study, comprising those with idiopathic pulmonary fibrosis (IPF) (n:8), autoimmune-related ILDs (auto-ILD) (n:4), and orphan diseases (O-ILD) (n:8), alongside five control subjects. Flow cytometry was employed to evaluate the Th to cytotoxic Tcell (CTL) ratio in BAL fluid, while cytopathological examination assessed macrophages, lymphocytes, and neutrophils. Quantitative real-time polymerase chain reaction was utilized to investigate the expressions in Th1, Th2, Th17, and regulatory T (Treg) cells. Results revealed elevated Th cell to CTL ratios across all patient groups compared to controls. Furthermore, upregulation of Th1, Th2, Th17, and T-cell factors was observed in all patient groups compared to controls. Interestingly, upregulation of CD28 and downregulation of CTLA-4 and PD-1 gene expression were consistent across all ILDs groups, highlighting potential immune dysregulation. This study provides a comprehensive exploration of molecular immunological mechanisms in ILDs patients, underscoring the dominance of Th2 and Th17 responses and revealing novel findings regarding the dysregulation of CD28, CTLA-4, and PD-1 expressions in ILDs for the first time.

NIR triggered polydopamine coated cerium dioxide nanozyme for ameliorating acute lung injury via enhanced ROS scavenging

Acute lung injury (ALI) is a life threatening disease in critically ill patients, and characterized by excessive reactive oxygen species (ROS) and inflammatory factors levels in the lung. Multiple evidences suggest that nanozyme with diversified catalytic capabilities plays a vital role in this fatal lung injury. At present, we developed a novel class of polydopamine (PDA) coated cerium dioxide (CeO2) nanozyme (Ce@P) that acts as the potent ROS scavenger for scavenging intracellular ROS and suppressing inflammatory responses against ALI. Herein, we aimed to identify that Ce@P combining with NIR irradiation could further strengthen its ROS scavenging capacity. Specifically, NIR triggered Ce@P exhibited the most potent antioxidant and anti-inflammatory behaviors in lipopolysaccharide (LPS) induced macrophages through decreasing the intracellular ROS levels, down-regulating the levels of TNF-α, IL-1β and IL-6, up-regulating the level of antioxidant cytokine (SOD-2), inducing M2 directional polarization (CD206 up-regulation), and increasing the expression level of HSP70. Besides, we performed intravenous (IV) injection of Ce@P in LPS induced ALI rat model, and found that it significantly accumulated in the lung tissue for 6 h after injection. It was also observed that Ce@P + NIR presented the superior behaviors of decreasing lung inflammation, alleviating diffuse alveolar damage, as well as promoting lung tissue repair. All in all, it has developed the strategy of using Ce@P combining with NIR irradiation for the synergistic enhanced treatment of ALI, which can serve as a promising therapeutic strategy for the clinical treatment of ROS derived diseases as well.

Enhanced anti-ovarian cancer efficacy with MSLN-chimeric antigen receptor T cells secreting anti-CD39 antibody.

e17553 Background: The effectiveness of chimeric antigen receptor (CAR) T cell therapy directed at mesothelin (MSLN) in solid tumors, such as ovarian cancer, is hindered by the immunosuppressive tumor microenvironment (TME), where adenosine plays a crucial role in impairing CAR-T cell function. Preclinical and clinical studies have demonstrated that targeting CD39, a pivotal enzyme catalyzing the conversion of extracellular ATP into adenosine, yields significant antitumor effects. Therefore, combining MSLN CAR-T with an anti-CD39 antibody emerges as a potential therapeutic strategy against cancer. Methods: We have previously designed OriA631, an antibody antagonist targeting CD39 in VHH format. Our preclinical investigation has highlighted the outstanding potential of OriA631, showcasing superior performance in binding affinity, effective inhibition of CD39 enzyme activity, and remarkable in vivo high anti-tumor efficacy. Subsequently, we undertook further engineering of MSLN CAR-T cells to secrete the VHH component of OriA631 (MSLN CAR-T-CD39-VHH) and systematically assessed its antitumor effects in NSG mice. Results: Our data reveals that MSLN CAR-T cells exhibit a significant upregulation of CD39 upon activation by ovarian cancer cells in both in vitro and in the tumor microenvironment (TME). To assess the combined effect of MSLN CAR-T with anti-CD39, B-NDG mice were subcutaneously inoculated with 3x10^6 ovarian cancer cells (SKOV3-MSLNhi-CD39hi). Subsequently, different T cells (1x10^7 cells/mouse) were injected when the tumor reached around 80 mm³, and the groups were divided into Mock T, MSLN CAR-T, MSLN CAR-T-CD39-VHH, and MSLN CAR-T in combination with locally injected CD39 Ab (OriA631). The data demonstrated that MSLN CAR-T inhibited tumor growth from approximately 1000mm³ (Mock T group) to 300mm³ at day 30 relative to T cell injection. Tumors in the MSLN CAR-T in combination with OriA631 group were around 100mm³, suggesting a significant combination anti-tumor efficacy of MSLN CAR-T in conjunction with CD39 targeting. Furthermore, MSLN CAR-T-CD39-VHH, where MSLN CAR-T cells secrete the VHH component of OriA631, completely eliminated the tumor at day 18 with no relapse during the experimental period. This result underscores the superior anti-tumor efficacy of MSLN CAR-T-CD39-VHH, with the high level of VHH component of CD39 Ab observed in the TME. Importantly, the low or undetectable level of VHH component of CD39 Ab in the serum also indicates the safety of the product. Conclusions: Our study introduces the innovative therapeutic agent MSLN CAR-T-CD39-VHH, showcasing superior anti-tumor efficacy and the potential for reduced antibody-associated side effects. These findings strongly endorse the application of MSLN CAR-T-CD39-VHH as a promising and impactful therapeutic approach for the treatment of ovarian cancer.

High-dimensional longitudinal immune profiling uncovers a dual role of the CXCL9/CXCR3, CXCL13/CXCR5, and CCL11/CCL3 axis in the coupling of immune-related adverse events to immune checkpoint inhibitor response.

2512 Background: Additional characterization is required to understand the immune network signatures and immunophenotypes that link immune-related adverse events (irAEs) to immune checkpoint inhibitor (ICI) therapy responses in cancer patients. Methods: A comprehensive immune profiling analysis was conducted on whole blood and peripheral blood mononuclear cells (PBMC) from 165 oncology patients with various irAEs, including colitis (n=64), myocarditis (n=19), pneumonitis (n=25), arthritis (n=24), and cytokine release syndrome CRS (n=33), and compared to a cohort of 219 cancer patients prior to ICI treatment. Results: Following ICI therapy on cycle 2 day 1 (C2D1), we observed significant increases in IL-6, CCL2, CXCL9, CXCL10, and CXCL13 levels preceding irAEs. CXCL9 was specifically upregulated during the development of colitis, arthritis, CRS, and myocarditis, while CXCL13 increased only in CRS, and CCL3 only in myocarditis and CRS, with no CCL11 upregulation in any irAE. High levels of CXCL9, high CCL11 and low CXCL13 strongly correlated with improved oncologic outcomes in different irAEs. Compared to other cluster, in the high CXCL9 cluster, 5-year overall survival (OS) was significantly improved (HR = 10.26 [95% CI, 1.27-82.82], p = 0.029) in colitis (n=20), in CRS (n=21) (HR = 6.45 [95% CI, 1.60-25.99], p = 0.009), and in arthritis. In the high CCL11 cluster, 5-year OS was significantly improved in pneumonitis (n=15), (HR = 4.04 [95% CI, 1.05-15.46]). Conversely, in myocarditis, the high CXCL13 cluster (n=9) was associated with decreased 2-year OS (HR = 6.05 [95% CI, 1.03-35.48], p = 0.046). It is important to note that the CXCL9-driven immunophenotypes were characterized by low IL-6. Notable observations include CD38 upregulation and large-scale activation of CD8 memory subsets during ICI therapy, concomitant with increased circulation of immature neutrophils at the onset of irAEs diagnosis. This event may increase the migration of TCRγδ, NK, DC, and CD8 cells to tumor sites through CXCR3/CXCL9 interactions. Conclusions: This study unravels intricate connections between immune activation and tumor response. CXCL9, CCL11, and CXCL13 emerge as pivotal biomarkers bridging the gap between ICI therapy effectiveness and distinct irAEs-associated immunophenotypes.

The Role of Fatty Acid Synthase in the Vascular Smooth Muscle Cell to Foam Cell Transition.

Vascular smooth muscle cells (VSMCs), in their contractile and differentiated state, are fundamental for maintaining vascular function. Upon exposure to cholesterol (CHO), VSMCs undergo dedifferentiation, adopting characteristics of foam cells-lipid-laden, macrophage-like cells pivotal in atherosclerotic plaque formation. CHO uptake by VSMCs leads to two primary pathways: ABCA1-mediated efflux or storage in lipid droplets as cholesterol esters (CEs). CE formation, involving the condensation of free CHO and fatty acids, is catalyzed by sterol O-acyltransferase 1 (SOAT1). The necessary fatty acids are synthesized by the lipogenic enzyme fatty acid synthase (FASN), which we found to be upregulated in atherosclerotic human coronary arteries. This observation led us to hypothesize that FASN-mediated fatty acid biosynthesis is crucial in the transformation of VSMCs into foam cells. Our study reveals that CHO treatment upregulates FASN in human aortic SMCs, concurrent with increased expression of CD68 and upregulation of KLF4, markers associated with the foam cell transition. Crucially, downregulation of FASN inhibits the CHO-induced upregulation of CD68 and KLF4 in VSMCs. Additionally, FASN-deficient VSMCs exhibit hindered lipid accumulation and an impaired transition to the foam cell phenotype following CHO exposure, while the addition of the fatty acid palmitate, the main FASN product, exacerbates this transition. FASN-deficient cells also show decreased SOAT1 expression and elevated ABCA1. Notably, similar effects are observed in KLF4-deficient cells. Our findings demonstrate that FASN plays an essential role in the CHO-induced upregulation of KLF4 and the VSMC to foam cell transition and suggest that targeting FASN could be a novel therapeutic strategy to regulate VSMC phenotypic modulation.

Upregulation of CD39 During Gout Attacks Promotes Spontaneous Remission of Acute Gouty Inflammation.

Gout is a self-limiting form of inflammatory arthropathy caused by the formation of urate crystals due to hyperuricemia. The resolution of gout involves the transition of proinflammatory M1-type macrophages to anti-inflammatory M2-type macrophages, as well as neutrophil-mediated extracellular trap (NET) formation. However, the underlying mechanisms of these changes are not clear. Studies have confirmed that high expression of CD39 on macrophages and neutrophils can trigger the polarization of macrophages from a proinflammatory state to an anti-inflammatory state. Recent studies have shown that the pathogenesis of gout involves extracellular ATP (eATP), and the synergistic effect of MSU and extracellular ATP can cause gout. CD39 is a kind of ATP hydrolysis enzyme that can degrade eATP, suggesting that CD39 may inhibit the aggravation of inflammation in gout and participate in the remission mechanism of gout. To confirm this hypothesis, using data mining and flow cytometry, we first found that CD39 expression was significantly upregulated on CD14 + monocytes and neutrophils in gout patients during the acute phase. Inhibition of CD39 by lentivirus or a CD39 inhibitor in acute gout models aggravated gouty arthritis and delayed gout remission. Apyrase, a functional analog of CD39, can significantly reduce the inflammatory response and promote gout remission in acute gout model mice. Our findings confirm that the upregulation of CD39 during gout flare-ups promotes spontaneous remission of acute gouty inflammation.

Epigenetic regulation of CD38/CD48 by KDM6A mediates NK cell response in multiple myeloma

Anti-CD38 monoclonal antibodies like Daratumumab (Dara) are effective in multiple myeloma (MM); however, drug resistance ultimately occurs and the mechanisms behind this are poorly understood. Here, we identify, via two in vitro genome-wide CRISPR screens probing Daratumumab resistance, KDM6A as an important regulator of sensitivity to Daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). Loss of KDM6A leads to increased levels of H3K27me3 on the promoter of CD38, resulting in a marked downregulation in CD38 expression, which may cause resistance to Daratumumab-mediated ADCC. Re-introducing CD38 does not reverse Daratumumab-mediated ADCC fully, which suggests that additional KDM6A targets, including CD48 which is also downregulated upon KDM6A loss, contribute to Daratumumab-mediated ADCC. Inhibition of H3K27me3 with an EZH2 inhibitor resulted in CD38 and CD48 upregulation and restored sensitivity to Daratumumab. These findings suggest KDM6A loss as a mechanism of Daratumumab resistance and lay down the proof of principle for the therapeutic application of EZH2 inhibitors, one of which is already FDA-approved, in improving MM responsiveness to Daratumumab.

Abstract WP318: Increased Synapse Elimination by Inflammatory Cells Contribute to Lon-Lasting Post-Stroke Memory Dysfunction in Aged Mice

Background and Purpose: Memory impairments are common after stroke. Memory dysfunction lasted longer in aged than in young mice, but the mechanisms are still unclear. Microglia maintain normal memory via removing excessive synapses and increase turnover of synaptic connections after stroke. Long-term memory deficits in aged mice may be associated with increased microglia in the brain. Hypothesis: Increased synapse removal by inflammatory cells contribute to the long-lasting post-stroke memory dysfunction of aged mice. Methods: Permanent distal middle cerebral artery occlusion (pMCAO) was induced in young and aged mice. The memory functions were analyzed weekly for 8 weeks by Y-maze test and at 8 weeks post stroke by novel objective recognition (NOR) test. Brain samples were collected 8 weeks after pMCAO induction. RNAseq analysis was performed to study gene expression profiles in peri-infarct area and hippocampus. Nissl staining was used to measure infarct volume and immunostaining was done to detect CD68 + and GFAP + cells and synapse in peri-infarct region and hippocampus. Results: RNAseq data shown up-regulation of CD68, GFAP, pro-inflammatory cytokines, immune response, microglia chemotaxins and phagocytosis genes; and down-regulation of synaptic assembly and transmission, synaptic plasticity, and long-term potentiation genes in aged mice at peri-infarct region and ipsilateral hippocampi. Aged mice had larger infarct volumes, more CD68 + and GFAP + cells in the peri-infarct region and ipsilateral hippocampi than young mice and had more CD68 + cells in the ipsilateral than the contralateral hippocampi. In Y-maze test, aged stroke mice made fewer spontaneous alternations from 3 to 8 weeks after pMCAO than young stroke and sham operated aged mice. In the NOR test, aged stroke mice spent less time on the novel objects than young stroke and sham aged mice. Increased synaptophysin positive CD68 + cells were detected in the hippocampi of aged stroke mice. Conclusions: Increase of synaptic elements by inflammatory cells contribute to the long-lasting post-stroke memory deficit in the aged mice, and reduction of neuroinflammation may improve their memory function.

Mononuclear cell composition and activation in blood and mucosal tissue of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic, inflammatory, antigen-driven disease of the esophagus. Tissue EoE pathology has previously been extensively characterized by novel transcriptomics and proteomic platforms, however the majority of surface marker determination and screening has been performed in blood due to mucosal tissue size limitations. While eosinophils, CD4+ T cells, mast cells and natural killer (NK) T cells were previously investigated in the context of EoE, an accurate picture of the composition of peripheral blood mononuclear cells (PBMC) and their activation is missing. In this study, we aimed to comprehensively analyze the composition of peripheral blood mononuclear cells and their activation using surface marker measurements with multicolor flow cytometry simultaneously in both blood and mucosal tissue of patients with active EoE, inactive EoE, patients with gastroesophageal reflux disease (GERD) and controls. Moreover, we set out to validate our data in co-cultures of PBMC with human primary esophageal epithelial cells and in a novel inducible mouse model of eosinophilic esophagitis, characterized by extensive IL-33 secretion in the esophagus. Our results indicate that specific PBMC populations are enriched, and that they alter their surface expression of activation markers in mucosal tissue of active EoE. In particular, we observed upregulation of the immunomodulatory molecule CD38 on CD4+ T cells and on myeloid cells in biopsies of active EoE. Moreover, we observed significant upregulation of PD-1 on CD4+ and myeloid cells, which was even more prominent after corticosteroid treatment. With co-culture experiments we could demonstrate that direct cell contact is needed for PD-1 upregulation on CD4+ T cells. Finally, we validated our findings of PD-1 and CD38 upregulation in an inducible mouse model of EoE. Herein we show significant alterations in the PBMC activation profile of patients with active EoE in comparison to inactive EoE, GERD and controls, which could have potential implications for treatment. To our knowledge, this study is the first of its kind expanding the multi-color flow cytometry approach in different patient groups using in vitro and in vivo translational models.

Neutrophil extracellular trap-induced intermediate monocytes trigger macrophage activation syndrome in adult-onset Still’s disease

BackgroundAdult-onset Still’s disease (AOSD) is a systemic autoinflammatory disease characterized by innate immune system activation, with a high risk for macrophage activation syndrome (MAS). MAS development is associated with monocyte/macrophage activation and cytokine storm. Monocytes consist of three different subsets, classical monocytes (CMs, CD14brightCD16 −), intermediate monocytes (IMs, CD14brightCD16 +), and non-classical monocytes (NCMs, CD14dimCD16 +), each has distinct roles in inflammatory regulation. However, the frequencies and regulatory mechanism of monocyte subsets in AOSD patients have not been identified.MethodsWe performed flow cytometry, RNA sequencing, phagocytosis analysis, and enzyme-linked immunosorbent assay to evaluate monocyte subsets, cell functions, and potential biomarkers. The effect of neutrophil extracellular traps (NETs) on monocytes was determined by evaluating mRNA levels of DNA sensors, surface CD16 expression, and inflammasome pathway activation.ResultsHigher proportions of intermediate monocytes (IMs) were identified in active AOSD patients. IMs displayed higher expression of CD80, CD86, HLA-DR, and CD163 than CMs and NCMs. CD163 upregulation was noted on AOSD IMs, accompanied by increased phagocytic activity and elevated cytokine/chemokine production, including IL-1β, IL-6, CCL8, and CXCL10. The frequencies of IMs were correlated with disease activity and higher in AOSD patients with MAS (AOSD-MAS). CCL8 and CXCL10 were highly expressed in RNA sequencing of monocytes from AOSD-MAS patients and plasma CXCL10 level could serve as a potential biomarker for AOSD-MAS. Moreover, DNA-sensing pathway was activated in monocytes from AOSD-MAS patients. Stimulation with NETs derived from AOSD induced DNA sensor expression, the expansion of IMs, and inflammasome pathway activation. These effects can be abrogated by DNase I treatment.ConclusionsOur results demonstrated that the proportions of IMs were elevated in AOSD and associated with MAS. The DNA component in NETs from AOSD plays an important role in the formation of IMs, shedding new light for the therapeutic target.

Sex-dimorphic neuroprotective effect of CD163 in an α-synuclein mouse model of Parkinson’s disease

Alpha-synuclein (α-syn) aggregation and immune activation represent hallmark pathological events in Parkinson’s disease (PD). The PD-associated immune response encompasses both brain and peripheral immune cells, although little is known about the immune proteins relevant for such a response. We propose that the upregulation of CD163 observed in blood monocytes and in the responsive microglia in PD patients is a protective mechanism in the disease. To investigate this, we used the PD model based on intrastriatal injections of murine α-syn pre-formed fibrils in CD163 knockout (KO) mice and wild-type littermates. CD163KO females revealed an impaired and differential early immune response to α-syn pathology as revealed by immunohistochemical and transcriptomic analysis. After 6 months, CD163KO females showed an exacerbated immune response and α-syn pathology, which ultimately led to dopaminergic neurodegeneration of greater magnitude. These findings support a sex-dimorphic neuroprotective role for CD163 during α-syn-induced neurodegeneration.

CD28 CAR T Cells for the Treatment of T Cell Malignancies

CONCLUSION While synthetic immunotherapy has been introduced into the standard of care treatment of patients with B-lineage malignancies, patients with T-lineage malignancies have not gained benefit from this novel approach, yet. The main reason is the challenging choice of target antigen in T-lineage malignancies such as pediatric T cell acute lymphoblastic leukemia (T-ALL). As activating mutations of CD28 have already been described in T cell malignancies, we set out to quantify surface expression of CD28 and other costimulatory molecules on primary pediatric T-ALL samples and explore the utility of this functionally relevant molecule as a novel target antigen. Bone marrow (BM) samples of pediatric T-ALL patients (n=54) at time of diagnosis and healthy control individuals (n=14) were collected. We quantified surface expression of CD28 and 25 additional co-stimulatory or co-inhibitory molecules by flow cytometry after gating on T cell precursors (living singlets, CD45 dim/SSC-A low and CD7 +). Subsequently, we generated a set of 20 CD28 directed second-generation chimeric antigen receptors (CARs) based on five different monoclonal antibodies with variation in hinge domain and light chain / heavy chain chronology. We analyzed specific CAR activity against CD28 expressing T-ALL cell lines after retroviral transduction into primary human T cells. Additional CD28 knockout (KO) by CRISPR/Cas9 could prevent T cell fratricide. CAR constructs were subjected to in vivo testing in a T-ALL NSG mouse model with transplantation of 7.5e4 CCRF-CEM cells transduced with firefly luciferase on day 0 and transfer of 2.5e6 CD28 KO T cells on day 3 harboring either of both CD28 CAR molecules. We used CD7 CAR T cells as positive control, CD19 CAR T cells and CD28 KO T cells without CAR as negative controls. We observed significant upregulation of CD28 on T-ALL leukemia when compared to healthy BM donors (Figure 1A, mean 68.8% vs. 3.7%, p=0.0002). We confirmed upregulation of CD28 in published T-ALL RNA-expression data sets. Interestingly, other co-stimulatory molecules such as CD127 were upregulated, too (mean 50.0% vs. 14.6%, p=0.0001), while co-inhibitory molecules such as CD160, TIGIT and TIM-3 were strongly downregulated (>10fold and p<0.0001 each). We hypothesized that this could imply a functional relevance of CD28 in T-ALL. Therefore, we performed co-culture assays of monocyte derived dendritic cells (CDs) expressing CD28 ligands CD80 and CD86 with different T-lineage leukemia cell lines. The presence of DCs significantly increased proliferation of CD28 + T-ALL cell lines under stress conditions. In order to test the feasibility of CD28 CAR T cells, we generated CD28 KO primary T cells without CAR. When tested in co-culture assays with leukemia cells and T cell engagers, CD28 KO T cells showed unchanged cytotoxic capacity and target-dependent activation illustrating that CD28 KO T cells retain short-term effector functions. Based on cytotoxic capacity and CAR T cell expansion, we identified two lead CD28 CAR candidates out of a pool of 20 CD28 CARs. After CD28 KO, CD28 CAR-T cells showed in vitro expansion comparable to CD19 CAR T cells. Next, we asked how CD28 CAR T cells compare to CD7 CAR T cells, which are currently evaluated clinical trials for T-ALL therapy. Therefore, we generated CD7 KO T cells that were subsequently transduced with a functional CD7 CAR. In vitro, both CD28 CAR T cells and CD7 CAR T cells showed killing of CD28 +CD7 + CCRF-CEM cells of >90% at an effector:target (E:T) ratio of 0.2:1, with CD7 being outperformed by one of the CD28 CARs while outperforming the other CD28 CAR at an E:T ratio of 0.04:1 (Figure 1B). Finally, we went on to validate the functionality of CD28 CAR T cells in vivo. We observed significantly prolonged survival of mice treated with CD28 CAR T cells and CD7 CAR T cells when compared to CD19 CAR T cells or control T cells. No difference in survival between CD7 and CD28 CAR T cell treated mice could be observed in two independent experiments. We identify CD28 as novel target antigen for pediatric T-ALL and provide evidence that CD28 is an immunotarget with functional relevance for T-ALL. We demonstrate the feasibility of CD28 CAR T cell generation and that these novel CAR T cells perform equally well as CD7 CAR T cells both in vitro and in vivo. Novel and functionally relevant CAR targets will facilitate clinical development of immunotherapy for T-lineage malignancies.

Development of Next Generation Antibody-Polymer-Drug Conjugates for Treatment of Multiple Myeloma

Background: Antibody-drug conjugates (ADCs) are an effective class of cancer therapeutics, but current ADCs face stability challenges, resulting in premature payload release and off-target toxicity. To overcome this, we developed a novel strategy using antibody-polymer-based drug conjugates (pADCs). Our pADCs have a number of advantages compared to currently FDA approved ADCs, including: 1) Increased drug payload without altering binding site numbers, ensuring maximum drug loading without affecting antigen and Fc binding; 2) Potential for synergistic immunotherapy and chemotherapy; 3) Improved pharmacokinetics and reduced immunogenicity with modification using water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, providing significant stealth effect; and 4) Enhanced stability under physiological conditions, reducing premature payload release and off-target toxicity. Panobinostat, a pan-HDAC (histone deacetylase) inhibitor, enhances CD38 expression and improves pADC efficiency against MM cells. Our hypothesis is that our pADC system induces strong cytotoxicity, further enhanced by panobinostat-induced CD38 upregulation, leading to more efficient treatment outcomes. Methods:mAbs decorated with cyclooctyne (DBCO) moieties in the hinge region were prepared in two steps: mAbs were reduced with tris(2-carboxyethyl)phosphine (TCEP), then thiol groups were capped with DBCO-PEG4-Mal. Azide-functionalized semitelechelic (ST) polymer-drug conjugates (ST-P-drug-N3) were synthesized via RAFT polymerization. Antibody-polymer-drug conjugates were prepared by CLICK chemistry. MM.1S and RPMI 8226 cell lines (ATCC) were used as the MM cell model. The cell internalization was revealed by confocal imaging. Primary MM cells were collected from the aspirate of eight patients with primary plasma cell leukemia and isolated via Ficoll centrifugation. CD38 upregulation was done by incubating with 10 or 25 nM panobinostat for 24 h and the apoptosis detection employed Annexin V and Propidium Iodide staining followed by flow cytometry analysis. Results: A series of CLICK-able mAbs and semitelechelic polymer-drug conjugates were synthesized. Among them, we firstly prepared Epirubicin (EPI)-based pADCs, such as DARA-P-EPI, ISA-P-EPI and DRO-P-EPI (Fig.1A). Unlike naked mAbs, pADCs can induce EPI concentration-dependent cytotoxicity. Surface calreticulin (CRT) was assessed and demonstrated the induction of EPI-dependent immunogenic cell death on MM.1S cells (Fig.1B). The pADC mechanism of action involves binding to cell surface antigens, internalization via endosomes, and subsequent cleavage of drugs at lysosomes by cathepsin B. This minimizes premature drug release and reduces off-target toxicity (Fig.1C). Ex vivo studies on patient MM cells revealed that pADCs induced significantly higher apoptosis compared to naked mAbs and free EPI, highlighting their efficacy (Fig.1D). CD38-based pADC's efficacy was enhanced by pretreatment with panobinostat (Fig.1F), which increased CD38 expression (Fig.1E). When incubating with NK cells and pADC, the number of target cells decreased significantly indicating the pADC preserve the original mAb ADCC functionality. Notably, co-treatment of RPMI8226 cells with anti-CD38 and GDC0980, a PI3k/mTOR inhibitor, significantly improved cytotoxicity, suggesting DARA's potential for chemo sensitization (Fig.1G). Conclusion: pADC's innovative design offers a high likelihood of successful clinical development and holds significant promise for MM therapy. Moreover, the unique mechanism provides potential to effectively overcome mAb and multiple drug resistance. Its facile nature allows for easy incorporation of multiple types of payloads into the ADC, making its synthesis highly scalable .

A CD38/CD28xCD3 Trispecific T-Cell Engager (TCE) As a Potentially Active Agent for the Treatment of Older Patients with Acute Myeloid Leukemia (AML)

Background: Refractory disease is common and the outcome of elderly AML patients unable to receive intensive chemotherapy with acceptable side effects remains dismal. Thus, the identification of effective and safer drugs in AML is warranted. Immunotherapeutic strategies have shown extraordinary efficacy for the treatment of hematological malignancies, such as anti-CD38 monoclonal antibodies (mAbs) in multiple myeloma. Indeed, CD38 could represent a potential therapeutic target for AML too, not only because it is expressed in AML blasts, but also because AML patients with heterogeneous or low CD38 expression could benefit from T-cell based immunotherapies that are less dependent of the density of the targeted antigen. Aims: To evaluate CD38 as a potential therapeutic target in AML, and to determine the mode of action and preclinical efficacy of isatuximab (an IgG1 anti-CD38 mAb) and SAR442257, which is a new CD38/CD28xCD3 trispecific TCE. Results: We evaluated CD38 expression in bone marrow blasts from 241 newly diagnosed elderly AML patients by flow cytometry. Overall, 99.2% of AML patients expressed CD38, 55.6% with homogeneous pattern and 44.6% with heterogeneous pattern. CD38 expression had no impact in the overall survival of AML patients. Interestingly, CD38 expression increased 2-fold in MRD positive leukemic blasts compared with patient-matched blasts at diagnosis (n=10). At 24h and up to 96h, isatuximab (70 nM) decreased CD38 surface level in 3 AML cell lines expressing low (KG-1), intermediate (MOLM-13) and high or similar (OCI-AML3) basal CD38 density as compared to AML patients. Isatuximab (70 nM) did not induce complement-dependent cytotoxicity; by contrast, we observed a trend to an increased antibody-dependent cellular phagocytosis and significant antibody-dependent cellular cytotoxicity (ADCC) only in OCI-AML3 cells in the presence of purified NK cells. In 17 primary samples from AML patients with homogeneous vs heterogeneous CD38 expression, isatuximab (70 nM) showed a median of 19% and 4.5% tumor-cell death, respectively. Altogether, these findings indicated an association between CD38 density and the mode of action triggered by isatuximab and its efficacy. As such, AML patients with heterogeneous CD38 expression are less likely to respond to isatuximab, but might benefit from TCE that are less dependent of the density of the targeted antigen. Contrary to isatuximab, the CD38/CD28xCD3 TCE did not induce a dose- and time-dependent decrease in CD38 surface levels in any AML cell line measured with a non-competing anti-CD38 antibody, regardless of the CD38 antigen density. When we cultured PBMCs from healthy adults (n=6) with MOLM-13 cells (E:T ratio of 10:1) in the presence of isatuximab or the CD38/CD28xCD3 TCE (700 pM), we observed a significantly higher tumor cell lysis at 48h induced by the latter. The same effect of the CD38/CD28xCD3 TCE was observed with purified T-, but not NK-cells from healthy adults (n=3). To identify the immune cells involved in tumor cell lysis, we confirmed that isatuximab only induced NK-cell activation, whereas the CD38/CD28xCD3 TCE induced both T- and NK-cell activation, measured as percentage of CD69+ cells. Co-culture of purified NK- or T-cells with MOLM-13 cells in the presence of the CD38/CD28xCD3 TCE demonstrated a T-cell dependent bystander activation of NK cells. Furthermore, isatuximab downregulated CD38 in a dose-dependent manner, especially in NK cells, while the CD38/CD28xCD3 TCE did not. Surprisingly, the CD38/CD28xCD3 TCE induced CD38 upregulation on T cells, indicating again T-cell activation. The efficacy of isatuximab and the CD38/CD28xCD3 TCE was analyzed in primary AML samples (n=8) and blast lysis was observed in two and three samples, respectively. Notably, two of the three CD38/CD28xCD3 TCE-responders were resistant to isatuximab. CD38/CD28xCD3 TCE-mediated lysis was independent of CD38 expression and induced no significant off-target toxicity. Conclusions: CD38 is widely present in blasts from older AML patients but nearly half show heterogeneous expression. While isatuximab-driven ADCC in AML cell lines and primary samples is dependent on CD38 density in tumor cells, the CD38/CD28xCD3 TCE exerted its anti-tumor efficacy regardless of CD38 density. Thus, AML patients expressing both high and low/heterogenous levels of CD38 could benefit from T cell based immunotherapeutic strategies targeting CD38.

NAD+ exhaustion by CD38 upregulation contributes to blood pressure elevation and vascular damage in hypertension

Hypertension is characterized by endothelial dysfunction and arterial stiffness, which contribute to the pathogenesis of atherosclerotic cardiovascular diseases. Nicotinamide adenine dinucleotide (NAD+) is an indispensable cofactor in all living cells that is involved in fundamental biological processes. However, in hypertensive patients, alterations in NAD+ levels and their relation with blood pressure (BP) elevation and vascular damage have not yet been studied. Here we reported that hypertensive patients exhibited lower NAD+ levels, as detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS), in both peripheral blood mononuclear cells (PBMCs) and aortas, which was parallel to vascular dysfunction. NAD+ boosting therapy with nicotinamide mononucleotide (NMN) supplement reduced BP and ameliorated vascular dysfunction in hypertensive patients (NCT04903210) and AngII-induced hypertensive mice. Upregulation of CD38 in endothelial cells led to endothelial NAD+ exhaustion by reducing NMN bioavailability. Pro-inflammatory macrophages infiltration and increase in IL-1β generation derived from pro-inflammatory macrophages resulted in higher CD38 expression by activating JAK1-STAT1 signaling pathway. CD38 KO, CD38 inhibitors treatment, or adeno-associated virus (AAV)-mediated endothelial CD38 knockdown lowered BP and improved vascular dysfunction in AngII-induced hypertensive mice. The present study demonstrated for the first time that endothelial CD38 activation and subsequently accelerated NAD+ degradation due to enhanced macrophage-derived IL-1β production was responsible for BP elevation and vascular damage in hypertension. NAD+ boosting therapy can be used as a novel therapeutic strategy for the management of hypertensive patients.